This invention relates to glycogen phosphorylase inhibitors, pharmaceutical compositions containing such inhibitors and the use of such inhibitors to treat diabetes, hyperglycemia, hypercholesterolemia, hypertension, hyperinsulinemias, hyperlipidemia, atherosclerosis and myocardial ischemia in mammals.
In spite of the early discovery of insulin and its subsequent widespread use in the treatment of diabetes, and the later discovery of and use of sulfonylureas (e.g. Chlorpropamide(trademark) (Pfizer), Tolbutamide(trademark) (Upjohn), Acetohexamide(trademark) (E. I. Lilly), Tolazamide(trademark) (Upjohn)) and biguanides (e.g. Phenformin(trademark) (Ciba Geigy), Mefformin(trademark) (G. D. Searle)) as oral hypoglycemic agents, the treatment of diabetes remains less than satisfactory. The use of insulin, necessary in about 10% of diabetic patients in which synthetic hypoglycemic agents are not effective (Type I diabetes, insulin dependent diabetes mellitus), requires multiple daily doses, usually by self injection. Determination of the proper dosage of insulin requires frequent estimations of the sugar in urine or blood. The administration of an excess dose of insulin causes hypoglycemia, with effects ranging from mild abnormalities in blood glucose to coma, or even death. Treatment of non-insulin dependent diabetes mellitus (Type II diabetes, NIDDM) usually consists of a combination of diet, exercise, oral agents, e.g. sulfonylureas, and in more severe cases, insulin. However, the clinically available hypoglycemics can have other side effects which limit their use. In any event, where one of these agents may fail in an individual case, another may succeed. A continuing need for hypoglycemic agents, which may have fewer side effects or succeed where others fail, is clearly evident.
Atherosclerosis, a disease of the arteries, is recognized to be the leading cause of death in the United States and Western Europe. The pathological sequence leading to atherosclerosis and occlusive heart disease is well known. The earliest stage in this sequence is the formation of xe2x80x9cfatty streaksxe2x80x9d in the carotid, coronary and cerebral arteries and in the aorta. These lesions are yellow in color due to the presence of lipid deposits found principally within smooth-muscle cells and in macrophages of the intima layer of the arteries and aorta. Further, it is postulated that most of the cholesterol found within the fatty streaks, in turn, give rise to development of the xe2x80x9cfibrous plaquexe2x80x9d, which consists of accumulated intimal smooth muscle cells laden with lipid and surrounded by extracellular lipid, collagen, elastin and proteoglyeans. The cells plus matrix form a fibrous cap that covers a deeper deposit of cell debris and more extra cellular lipid. The lipid is primarily free and esterified cholesterol. The fibrous plaque forms slowly, and is likely in time to become calcified and necrotic, advancing to the xe2x80x9ccomplicated lesionxe2x80x9d which accounts for the arterial occlusion and tendency toward mural thrombosis and arterial muscle spasm that characterize advanced atherosclerosis.
Epidemiological evidence has firmly established hyperlipidemia as a primary risk factor in causing cardiovascular disease (CVD) due to atherosclerosis. In recent years, leaders of the medical profession have placed renewed emphasis on lowering plasma cholesterol levels, and low density lipoprotein cholesterol in particular, as an essential step in prevention of CVD. The upper limits of xe2x80x9cnormalxe2x80x9d are now known to be significantly lower than heretofore appreciated. As a result, large segments of Western populations are now realized to be at particular high risk. Such independent risk factors include glucose intolerance, left ventricular hypertrophy, hypertension, and being of the male sex. Cardiovascular disease is especially prevalent among diabetic subjects, at least in part because of the existence of multiple independent risk factors in this population. Successful treatment of hyperlipidemia in the general population, and in diabetic subjects in particular, is therefore of exceptional medical importance.
Hypertension (or high blood pressure) is a condition which occurs in the human population as a secondary symptom to various other disorders such as renal artery stenosis, pheochromocytoma or endocrine disorders. However, hypertension is also evidenced in many patients in whom the causative agent or disorder is unknown. While such xe2x80x9cessentialxe2x80x9d hypertension is often associated with disorders such as obesity, diabetes and hypertriglyceridemia, the relationship between these disorders has not been elucidated. Additionally, many patients display the symptoms of high blood pressure in the complete absence of any other signs of disease or disorder.
It is known that hypertension can directly lead to heart failure, renal failure and stroke (brain hemorrhaging). These conditions are capable of causing short-term death in a patient. Hypertension can also contribute to the development of atherosclerosis and coronary disease. These conditions gradually weaken a patient and can led to long-term death.
The exact cause of essential hypertension is unknown, though a number of factors are believed to contribute to the onset of the disease. Among such factors are stress, uncontrolled emotions, unregulated hormone release (the renin, angiotensin, aldosterone system), excessive salt and water due to kidney malfunction, wall thickening and hypertrophy of the vasculature resulting in constricted blood vessels and genetic factors.
The treatment of essential hypertension has been undertaken bearing the foregoing factors in mind. Thus a broad range of beta-blockers, vasoconstrictors, angiotensin converting enzyme inhibitors and the like have been developed and marketed as antihypertensives. The treatment of hypertension utilizing these compounds has proven beneficial in the prevention of short-interval deaths such as heart failure, renal failure and brain hemorrhaging. However, the development of atherosclerosis or heart disease due to hypertension over a long period of time remains a problem. This implies that although high blood pressure is being reduced, the underlying cause of essential hypertension is not responding to this treatment.
Hypertension has been associated with elevated blood insulin levels, a condition known as hyperinsulinemia. Insulin, a peptide hormone whose primary actions are to promote glucose utilization, protein synthesis and the formation and storage of neutral lipids, also acts to promote vascular cell growth and increase renal sodium retention, among other things. These latter functions can be accomplished without affecting glucose levels and are known causes of hypertension. Peripheral vasculature growth, for example, can cause constriction of peripheral capillaries; while sodium retention Sincreases blood volume. Thus, the lowering of insulin levels in hyperinsulinemics can prevent abnormal vascular growth and renal sodium retention caused by high insulin levels and thereby alleviate hypertension.
Cardiac hypertrophy is a significant risk factor in the development of sudden death, myocardial infarction, and congestive heart failure. These cardiac events are due, at least in part, to increased susceptibility to myocardial injury after ischemia and reperfusion which can occur in out-patient as well as perioperative settings. There is an unmet medical need to prevent or minimize adverse myocardial perioperative outcomes, particularly perioperative myocardial infarction. Both non-cardiac and cardiac surgery are associated with substantial risks for myocardial infarction or death. Some 7 million patients undergoing non-cardiac surgery are considered to be at risk, with incidences of perioperative death and serious cardiac complications as high as 20-25% in some series. In addition, of the 400,000 patients undergoing coronary by-pass surgery annually, perioperative myocardial infarction is estimated to occur in 5% and death in 1-2%. There is currently no drug therapy in this area which reduces damage to cardiac tissue from perioperative myocardial ischemia or enhances cardiac resistance to ischemic episodes. Such a therapy is anticipated to be life-saving and reduce hospitalizations, enhance quality of life and reduce overall health care costs of high risk patients.
Hepatic glucose production is an important target for NIDDM therapy. The liver is the major regulator of plasma glucose levels in the post absorptive (fasted) state, and the rate of hepatic glucose production in NIDDM patients is significantly elevated relative to normal individuals. Likewise, in the postprandial (fed) state, where the liver has a proportionately smaller role in the total plasma glucose supply, hepatic glucose production is abnormally high in NIDDM patients.
Glycogenolysis is an important target for interruption of hepatic glucose production. The liver produces glucose by glycogenolysis (breakdown of the glucose polymer glycogen) and gluconeogenesis (synthesis of glucose from 2- and 3-carbon precursors). Several lines of evidence indicate that glycogenolysis may make an important contribution to hepatic glucose output in NIDDM. First, in normal post absorptive man, up to 75% of hepatic glucose production is estimated to result from glycogenolysis. Second, patients having liver glycogen storage diseases, including Hers"" disease (glycogen phosphorylase deficiency), display episodic hypoglycemia. These observations suggest that glycogenolysis may be a significant process for hepatic glucose production.
Glycogenolysis is catalyzed in liver, muscle, and brain by tissue-specific isoforms of the enzyme glycogen phosphorylase. This enzyme cleaves the glycogen macromolecule to release glucose-1-phosphate and a new shortened glycogen macromolecule. Two types of glycogen phosphorylase inhibitors have been reported to date: glucose and glucose analogs [Martin, J. L. et al. Biochemistry 1991, 30, 10101] and caffeine and other purine analogs [Kasvinsky, P. J. et al. J. Biol. Chem. 1978, 253, 3343-3351 and 9102-9106]. These compounds, and glycogen phosphorylase inhibitors in general, have been postulated to be of potential use for the treatment of NIDDM by decreasing hepatic glucose production and lowering glycemia. [Blundell, T. B. et al. Diabetologia 1992, 35, Suppl. 2, 569-576 and Martin et al. Biochemistry 1991, 30, 10101].
The mechanism(s) responsible for the myocardial injury observed after ischemia and reperfusion is not fully understood. It has been reported (M. F. Allard, et al. Am. J. Physiol. 267, H66-H74, 1994) that xe2x80x9cpre ischemic glycogen reduction . . . is associated with improved post ischemic left ventricular functional recovery in hypertrophied rat heartsxe2x80x9d.
Thus, although there are a variety of hyperglycemia, hypercholesterolemia, hypertension, hyperlipidemia, atherosclerosis and myocardial ischemia therapies there is a continuing need and a continuing search in this field of art for alternative therapies.
This invention is directed to glycogen phosphorylase inhibitor compounds of Formula I useful for the treatment of diabetes, hyperglycemia, hypercholesterolemia, hypertension, hyperinsulinemia, hyperlipidemia, atherosclerosis and myocardial ischemia.
The compounds of this invention have the Formula I 
and the pharmaceutically acceptable salts and prodrugs thereof wherein
the dotted line ( - - - ) is an optional bond;
A is xe2x80x94C(H)xe2x95x90, xe2x80x94C((C1-C4)alkyl)= or xe2x80x94C(halo)= when the dotted line ( - - - ) is a bond, or A is methylene or xe2x80x94CH((C1-C4)alkyl)xe2x80x94 when the dotted line ( - - - ) is not a bond;
R1, R10 or R11 are each independently H, halo, 4-, 6- or 7-nitro, cyano, (C1-C4)alkyl, (C1-C4)alkoxy, fluoromethyl, difluoromethyl or trifluoromethyl;
R2 is H;
R3 is H or (C1-C5)alkyl;
R4 is H, methyl, ethyl, n-propyl, hydroxy(C1-C3)alkyl, (C1-C3)alkoxy(C1-C3)alkyl, phenyl(C1-C4)alkyl, phenylhydroxy(C1-C4)alkyl, phenyl(C1-C4)alkoxy(C1-C4)alkyl, thien-2- or 3-yl(C1-C4)alkyl or fur-2- or -3-yl(C1-C4)alkyl wherein said R4 rings are mono-, di- or tri-substituted independently on carbon with H, halo, (C1-C4)alkyl, (C1-C4)alkoxy, trifluoromethyl, hydroxy, amino or cyano; or
R4 is pyrid-2-, -3- or 4-yl(C1-C4)alkyl, thiazol-2-, -4- or -5-yl(C1-C4)alkyl, imidazol-1-, -2-, -4- or -5-yl(C1-C4)alkyl, pyrrol-2- or -3-yl(C1-C4)alkyl, oxazol-2-, -4- or -5-yl-(C1-C4)alkyl, pyrazol-3-, -4- or -6-yl(C1-C4)alkyl, isoxazol-3-, -4- or -5-yl(C1-C4)alkyl, isothiazol-3-, -4- or -5-yl(C1-C4)alkyl, pyridazin-3- or -4-yl-(C1-C4)alkyl, pyrimidin-2-, -4-, -5- or 6-yl(C1-C4)alkyl, pyrazin-2- or -3-yl(C1-C4)alkyl or 1,3,5triazin-2-yl(C1-C4)alkyl, wherein said preceding R4 heterocycles are optionally mono- or di-substituted independently with halo, trifluoromethyl, (C1-C4)alkyl, (C1-C4)alkoxy, amino or hydroxy and said mono- or di-substituents are bonded to carbon;
R5 is H, hydroxy, fluoro, (C1-C5)alkyl, (C1-C5)alkoxy, (C1-C6)alkanoyl, amino(C1-C4)alkoxy, mono-N- or di-N,N-(C1-C4)alkylamino(C1-C4)alkoxy, carboxy(C1-C4)alkoxy, (C1-C5)alkoxy-carbonyl(C1-C4)alkoxy, benzyloxycarbonyl(C1-C4)alkoxy, or carbonyloxy wherein said carbonyloxy is carbonxe2x80x94carbon linked with phenyl, thiazolyl, imidazolyl, 1H-indolyl, turyl, pyrrolyl, oxazolyl, pyrazolyl, isoxazolyl, isothiazolyl, pyridazinyl, pyrimidinyl, pyrazinyl or 1,3,5-triazinyl and wherein said preceding R5 rings are optionally mono-substituted with halo, (C1-C4)alkyl, (C1-C4)alkoxy, hydroxy, amino or trifluoromethyl and said mono-substituents are bonded to carbon;
R7 is H, fluoro or (C1-C5)alkyl; or
R5 and R7 can be taken together to be oxo;
R6 is carboxy, (C1-C8)alkoxycarbonyl, C(O)NR8R9 or C(O)R12, wherein
R8 is H, (C1-C3)alkyl, hydroxy or (C1-C3)alkoxy; and
R9 is H, (C1-C8)alkyl, hydroxy, (C1-C8)alkoxy, methylene-perfluorinated(C1-C8)alkyl, phenyl, pyridyl, thienyl, furyl, pyrrolyl, pyrrolidinyl, oxazolyl, thiazolyl, imidazolyl, pyrazolyl, pyrazolinyl, pyrazolidinyl, isoxazolyl, isothiazolyl, pyranyl, piperidinyl, morpholinyl, pyridazinyl, pyrimidinyl, pyrazinyl, piperazinyl or 1,3,5-triazinyl wherein said preceding R9 rings are carbon-nitrogen linked; or
R9 is mono-, di- or tri-substituted (C1-C5)alkyl, wherein said substituents are independently H, hydroxy, amino, mono-N- or di-N,N-(C1-C6)alkylamino; or
R9 is mono- or di-substituted (C1-C5)alkyl, wherein said substituents are independently phenyl, pyridyl, furyl, pyrrolyl, pyrrolidinyl, oxazolyl, thiazolyl, imidazolyl, pyrazolyl, pyrazolinyl, pyrazolidinyl, isoxazolyl, isothiazolyl, pyranyl, pyridinyl, piperidinyl, morpholinyl, pyridazinyl, pyrimidinyl, pyrazinyl, piperazinyl or 1,3,5-triazinyl
wherein the nonaromatic nitrogen-containing R9 rings are optionally mono-substituted on nitrogen with (C1-C6)alkyl, benzyl, benzoyl or (C1-C6)alkoxycarbonyl and wherein the R9 rings are optionally mono-substituted on carbon with halo, (C1-C4)alkyl, (C1-C4)alkoxy, hydroxy, amino, or mono-N- and di-N,N(C1-C5)alkylamino provided that no quaternized nitrogen is included and there are no nitrogen-oxygen, nitrogenxe2x80x94nitrogen or nitrogen-halo bonds;
R12 is piperazin-1-yl, 4-(C1-C4)alkylpiperazin-1-yl, 4-formylpiperazin-1-yl, morpholino, thiomorpholino, 1-oxothiomorpholino, 1,1-dioxo-thiomorpholino, thiazolidin-3-yl, 1-oxo-thiazolidin-3-yl, 1,1-dioxo-thiazolidin-3-yl, 2-(C1-C6)alkoxycarbonylpyrrolidin-1-yl, oxazolidin-3-yl or 2(R)-hydroxymethylpyrrolidin-1-yl; or
R12 is 3- and/or 4-mono- or di-substituted oxazetidin-2-yl, 2-, -4-, and/or 5-mono- or di-substituted oxazolidin-3-yl, 2-, 4-, and/or 5- mono- or di- substituted thiazolidin-3-yl, 2-, 4-, and/or 5- mono- or di- substituted 1-oxothiazolidin-3-yl, 2-, 4-, and/or 5- mono- or di- substituted 1,1-dioxothiazolidin-3-yl, 3- and/or 4-, mono- or di-substituted pyrrolidin-1-yl, 3-, 4- and/or 5-, mono-, di- or tri-substituted piperidin-1-yl, 3-, 4-, and/or 5- mono-, di-, or tri-substituted piperazin-1-yl, 3-substituted azetidin-1-yl, 4- and/or 5-, mono- or di-substituted 1,2-oxazinan-2-yl, 3- and/or 4-mono- or di-substituted pyrazolidin-1-yl, 4- and/or 5-, mono- or di-substituted isoxazolidin-2-yl, 4- and/or 5-, mono- and/or di-substituted isothiazolidin-2-yl wherein said R12 substituents are independently H, halo, (C1-C5)-alkyl, hydroxy, amino, mono-N- or di-N,N-(C1-C5)alkylamino, formyl, oxo, hydroxyimino, (C1-C5)alkoxy, carboxy, carbamoyl, mono-N- or di-N,N-(C1-C4)alkylcarbamoyl, (C1-C4)alkoxyimino, (C1-C4)alkoxymethoxy, (C1-C6)alkoxycarbonyl, carboxy(C1-C5)alkyl or hydroxy(C1-C5)alkyl;
with the proviso that if R4 is H, methyl, ethyl or n-propyl R5 is OH;
with the proviso that if R5 and R7 are H, then R4 is not H, methyl, ethyl, n-propyl, hydroxy(C1-C3)alkyl or (C1-C3)alkoxy(C1-C3)alkyl and R, is C(O)NR8R9, C(O)R12 or (C1-C4)alkoxycarbonyl.
A first group of preferred compounds of Formula I consists of those compounds wherein
R1 is 5-H, 5-halo, 5-methyl or 5-cyano;
R10 and R11 are each independently H or halo;
A is xe2x80x94C(H)xe2x95x90;
R2 and R3 are H;
R4 is phenyl(C1-C2)alkyl wherein said phenyl groups are mono-, di- or tri-substituted independently with H or halo or mono- or di- substituted independently with H, halo, (C1-C4)alkyl, (C1-C4)alkoxy, trifluoromethyl, hydroxy, amino or cyano; or
R4 is thien-2- or -3-yl(C1-C2)alkyl, pyrid-2-, -3- or 4-yl(C1-C2)alkyl, thiazol-2-, -4- or -5-yl(C1-C2)alkyl, imidazol-1-, -2-, -4- or -5-yl(C1-C2)alkyl, fur-2- or -3-yl(C1-C2)alkyl, pyrrol-2- or -3-yl(C1-C2)alkyl, oxazol-2-, -4- or -5-yl-(C1-C2)alkyl, pyrazol-3-, -4- or -5-yl(C1-C2)alkyl, isoxazol-3-, -4- or -5-yl(C1-C2)alkyl wherein said preceding R4 heterocycles are optionally mono- or di-substituted independently with halo, trifluoromethyl, (C1-C4)alkyl, (C1-C4)alkoxy, amino or hydroxy and said mono- or di-substituents are bonded to carbon;
R5 is hydroxy;
R6 is C(O)NR8R9 or C(O)R12; and
R7 is H.
Within the above first group of preferred compounds of Formula I is a first group of especially preferred compounds wherein
the carbon atom a has (S) stereochemistry;
the carbon atom b has (R) stereochemistry;
R4 is phenyl(C1-C2)alkyl, thien-2-yl-(C1-C2)alkyl, thien-3-yl-(C1-C2)alkyl, fur-2-yl-(C1-C2)alkyl or fur-3-yl-(C1-C2)alkyl wherein said rings are mono- or di- substituted independently with H or fluoro;
R6 is C(O)NR8R9;
R8 is (C1-C3)alkyl, hydroxy or (C1-C3)alkoxy; and
R9 is H, (C1-C8)alkyl, hydroxy, hydroxy(C1-C6)alkyl, (C1-C8)alkoxy, pyridyl, morpholinyl, piperazinyl, pyrrolidinyl, piperidinyl, imidazolyl or thiazolyl or (C1-C4)alkyl mono-substituted with pyridyl, morpholinyl, piperazinyl, pyrrolidinyl, piperidinyl, imidazolyl or thiazolyl.
Within the above first group of especially preferred compounds are the particularly preferred compounds
5-Chloro-1H-indole-2-carboxylic acid [(1S)-((R)-hydroxy-dimethylcarbamoyl-methyl)-2-phenyl-ethyl]-amide,
5,6-Dichloro-1H-indole-2-carboxylic acid {(1S)-[(R)-hydroxy-(methoxy-methyl-carbamoyl)-methyl]-2-phenyl-ethyl}-amide,
5-Chloro-1H-indole-2-carboxylic acid {(1S)-[(R)-hydroxy-(methoxy-methyl-carbamoyl)-methyl]-2-phenyl-ethyl}-amide,
5-Chloro-1H-indole-2-carboxylic acid ((1S)-{(R)-hydroxy-[(2-hydroxy-ethyl)-methyl-carbamoyl]-methyl}-2-phenyl-ethyl)-amide,
5-Chloro-1H-indole-2-carboxylic acid {(1S)-[(R)-hydroxy-(methyl-pyridin-2-yl-carbamoyl)-methyl]-2-phenyl-ethyl}-amide or
5-Chloro-1H-indole-2-carboxylic acid ((1S)-{(R)-hydroxy-[methyl-(2-pyridin-2-yl-ethyl)-carbamoyl]-methyl}-2-phenyl-ethyl)-amide.
Within the above first group of especially preferred compounds are the compounds wherein
a.
R1 is 5-chloro;
R10 and R11 are H;
R4 is benzyl;
R8 is methyl; and
R9 is methyl;
b.
R1 is 5-chloro;
R11 is H;
R10 is 6-chloro;
R4 is benzyl;
R8 is methyl; and
R9 is methoxy;
c.
R1 is 5-chloro;
R10 and R11 are H;
R4 is benzyl;
R8 is methyl; and
R9 is methoxy;
d.
R1 is 5-chloro;
R10 and R11 are H;
R4 is benzyl;
R8 is methyl; and
R9 is 2-(hydroxy)ethyl;
e.
R1 is 5-chloro;
R10 and R11 are H;
R4 is benzyl;
R8 is methyl; and
R9 is pyridin-2-yl; and
f.
R1 is 5-chloro;
R10 and R11 are H;
R4 is benzyl;
R8 is methyl; and
R9 is 2-(pyridin-2-yl)ethyl.
Within the above first group of preferred compounds of Formula I is a second group of especially preferred compounds wherein the carbon atom a is (S) stereochemistry;
the carbon atom b is (R) stereochemistry;
R4 is phenyl(C1-C2)alkyl, thien-2-yl-(C1-C2)alkyl, thien-3-yl-(C1-C2)alkyl, fur-2-yl-(C1-C2)alkyl or fur-3-yl-(C1-C2)alkyl wherein said rings are mono- or di- substituted independently with H or fluoro;
R6 is C(O)R12; and
R12 is morpholino, 4-(C1-C4)alkylpiperazin-1-yl, 3-substituted azetidin-1-yl, 3- and/or 4-, mono- or di-substituted pyrrolidin-1-yl, 4- and/or 5- mono- or di-substituted isoxazolidin-2-yl, 4- and/or 5-, mono- or di-substituted 1,2-oxazinan-2-yl wherein said substituents are each independently H, halo, hydroxy, amino, mono-N- or di-N,N-(C1-C6)alkylamino, oxo, hydroxyimino or alkoxy.
Within the above second group of especially preferred compounds are the particularly preferred compounds
5-Chloro-1H-indole-2-carboxylic acid [(1S)-benzyl-(2R)-hydroxy-3-(4-methyl-piperazin-1-yl)-3-oxo-propyl]-amide hydrochloride,
5-Chloro-1H-indole-2-carboxylic acid [(1S)-benzyl-(2R)-hydroxy-3-(3-hydroxy-azetidin-1-yl)-3-oxo-propyl]-amide,
5-Chloro-1H-indole-2-carboxylic acid ((1S)-benzyl-(2R)-hydroxy-3-isoxazolidin-2-yl-3-oxo-propyl)-amide,
5-Chloro-1H-indole-2-carboxylic acid ((1S)-benzyl-(2R)-hydroxy-3-[1,2]-oxazinan-2-yl-3-oxo-propyl)-amide,
5-Chloro-1H-indole-2-carboxylic acid [(1S)-benzyl-(2R)-hydroxy-3-((3S)-hydroxy-pyrrolidin-1-yl)-3-oxo-propyl]-amide,
5-Chloro-1H-indole-2-carboxylic acid [(1S)-benzyl-3-((3S,4S)-dihydroxy-pyrrolidin-1-yl)-(2R)-hydroxy-3-oxo-propyl]-amide,
5-Chloro-1H-indole-2-carboxylic acid [(1S)-benzyl-3-((3R,4S)-dihydroxy-pyrrolidin-1-yl)-(2R)-hydroxy-3-oxo-propyl]-amide or
5-Chloro-1H-indole-2-carboxylic acid ((1S)-benzyl-(2R)-hydroxy-3-morpholin-4-yl-3-oxo-propyl)-amide.
Within the above second group of especially preferred compounds are the compounds wherein
a.
R1 is 5-chloro;
R10 and R11 are H;
R4 is benzyl; and
R12 is 4-methylpiperazin-1-yl;
b.
R1 is 5-chloro;
R10 and R11 are H;
R4 is benzyl; and
R12 is 3-hydroxyazetidin-1-yl;
c.
R1 is 5-chloro;
R10 and R11 are H;
R4 is benzyl; and
R12 is isoxazolidin-2-yl;
d.
R1 is 5-chloro;
R10 and R11 are H;
R4 is benzyl; and
R12 is (1,2)-oxazinan-2-yl;
e.
R1 is 5-chloro;
R10 and R11 are H;
R4 is benzyl; and
R12 is 3(S)-hydroxypyrrolidin-1-yl;
f.
R1 is 5-chloro;
R10 and R11 are H;
R4 is benzyl; and
R12 is (3S,4S)-dihydroxypyrrolidin-1-yl;
g.
R1 is 5-chloro;
R10 and R11 are H;
R4 is benzyl; and
R12 is (3R,4S)-dihydroxypyrrolidin-1-yl; and
h.
R1 is 5-chloro;
R10 and R11 are H;
R4 is benzyl; and
R12 is morpholino.
A second group of preferred compounds of Formula I consists of those compounds wherein
R1 is H, halo, methyl or cyano;
R10 and R11 are each independently H or halo;
A is xe2x80x94C(H)xe2x95x90;
R2 and R3 are H;
R4 is phenyl(C1-C2)alkyl wherein said phenyl groups are mono-, di- or tri-substituted independently with H or halo or mono- or di- substituted independently with H, halo, (C1-C4)alkyl, (C1-C4)alkoxy, trifluoromethyl, hydroxy, amino or cyano; or
R4 is thien-2- or -3-yl(C1-C2)alkyl, pyrid-2-, -3- or -4yl(C1-C2)alkyl, thiazol-2-, -4- or -5-yl(C1-C2)alkyl, imidazol-1-, -2-, -4- or -5-yl(C1-C2)alkyl, fur-2- or -3-yl(C1-C2)alkyl, pyrrol-2- or -3-yl(C1-C2)alkyl, oxazol-2-, -4-or -5-yl-(C1-C2)alkyl, pyrazol-3-, -4- or -5-yl(C1-C2)alkyl, isoxazol-3-, -4- or -5-yl(C1-C2)alkyl wherein said preceding R4 heterocycles are optionally mono- or di-substituted independently with halo, trifluoromethyl, (C1-C4)alkyl, (C1-C4)alkoxy, amino or hydroxy and said mono- or di-substituents are bonded to carbon;
R5 is hydroxy;
R6 is carboxy or (C1-C8)alkoxycarbonyl; and
R7 is H, fluoro or (C1-C6)alkyl.
Within the second group of preferred compounds of Formula I is a group of especially preferred compounds wherein
the carbon atom a is (S) stereochemistry;
the carbon atom b is (R) stereochemistry;
R4 is phenyl(C1-C2)alkyl, thien-2-yl-(C1-C2)alkyl, thien-3-yl-(C1-C2)alkyl, fur-2-yl-(C1-C2)alkyl or fur-3-yl-(C1-C2)alkyl wherein said rings are mono- or di- substituted independently with H or fluoro;
R10 and R11 are H;
R6 is carboxy; and
R7 is H.
Preferred within the immediately preceding group is a compound wherein
R1 is 5-chloro;
R10 and R11 are H; and
R4 is benzyl.
A third group of preferred compounds of Formula I consists of those compounds wherein
R1 is H, halo, methyl or cyano;
R10 and R11 are each independently H or halo;
A is xe2x80x94C(H)xe2x95x90;
R2 and R3 are H;
R4 is phenyl(C1-C2)alkyl wherein said phenyl groups are mono-, di- or tri-substituted independently with H or halo or mono- or di- substituted independently with H, halo, (C1-C4)alkyl, (C1-C4)alkoxy, trifluoromethyl, hydroxy, amino or cyano; or
R4 is thien-2- or -3-yl(C1-C2)alkyl, pyrid-2-, -3- or 4-yl(C1-C2)alkyl, thiazol-2-, -4- or -5-yl(C1-C2)alkyl, imidazol-1-, -2-, -4- or -5-yl(C1-C2)alkyl, fur-2- or -3-yl(C1-C2)alkyl, pyrrol-2- or -3-yl(C1-C2)alkyl, oxazol-2-, -4- or -5-yl-(C1-C2)alkyl, pyrazol-3-, -4- or -5-yl(C1-C2)alkyl, isoxazol-3-, -4- or -5-yl(C1-C2)alkyl wherein said preceding R4 heterocycles are optionally mono- or di-substituted independently with halo, trifluoromethyl, (C1-C4)alkyl, (C1-C4)alkoxy, amino or hydroxy and said mono- or di-substituents are bonded to carbon;
R5 is fluoro, (C1-C4)alkyl, (C1-C5)alkoxy, amino(C1-C4)alkoxy, mono-N- or di-N,N-(C1-C4)alkylamino(C1-C4)alkoxy, carboxy(C1-C4)alkoxy, (C1-C5)alkoxy-carbonyl(C1-C4)alkoxy, benzyloxycarbonyl(C1-C4)alkoxy;
R6 is carboxy or (C1-C8)alkoxycarbonyl; and
R7 is H, fluoro or (C1-C6)alkyl.
A fourth group of preferred compounds of Formula I consists of those compounds wherein
R1 is H, halo, methyl or cyano;
R10 and R11 are each independently H or halo;
A is xe2x80x94C(H)xe2x95x90;
R2 and R3 are H;
R4 is phenyl(C1-C2)alkyl wherein said phenyl groups are mono-, di- or tri-substituted independently with H or halo or mono- or di- substituted independently with H, halo, ((C1-C4)alkyl, (C1-C4)alkoxy, trifluoromethyl, hydroxy, amino or cyano; or
R4 is thien-2- or -3-yl(C1-C2)alkyl, pyrid-2-, -3- or 4-yl(C1-C2)alkyl, thiazol-2-, -4- or -5-yl(C1-C2)alkyl, imidazol-1-, -2-, -4- or -5-yl(C1-C2)alkyl, fur-2- or -3-yl(C1-C2)alkyl, pyrrol-2- or -3-yl(C1-C2)alkyl, oxazol-2-, -4- or -5-yl-(C1-C2)alkyl, pyrazol-3-, -4- or -5-yl(C1-C2)alkyl, isoxazol-3-, -4- or -5-yl(C1-C2)alkyl wherein said preceding R4 heterocycles are optionally mono- or di-substituted independently with halo, trifluoromethyl, (C1-C4)alkyl, (C1-C4)alkoxy, amino or hydroxy and said mono- or di-substituents are bonded to carbon;
R5 is fluoro, (C1-C4)alkyl, (C1-C5)alkoxy, amino(C1-C4)alkoxy, mono-N- or di-N,N-(C1-C4)alkylamino(C1-C4)alkoxy, carboxy(C1-C4)alkoxy, (C1-C5)alkoxy-carbonyl(C1-C4)alkoxy, benzyloxycarbonyl(C1-C4)alkoxy;
R6 is C(O)NR8R9 or C(O)R12; and
R7 is H, fluoro or (C1-C6)alkyl.
Yet another aspect of this invention is directed to a method for treating a glycogen phosphorylase dependent disease or condition in a mammal by administering to a mammal suffering from a glycogen phosphorylase dependent disease or condition a glycogen phosphorylase dependent disease or condition treating amount of a Formula I compound.
Yet another aspect of this invention is directed to a method for treating hyperglycemia in a mammal by administering to a mammal suffering from hyperglycemia a hyperglycemia treating amount of a Formula I compound.
Yet another aspect of this invention is directed to a method for treating diabetes in a mammal by administering to a mammal suffering from diabetes a diabetes treating amount of a Formula I compound.
Yet another aspect of this invention is directed to a method for treating hypercholesterolemia in a mammal by administering to a mammal suffering from hypercholesterolemia a hypercholesterolemia treating amount of a Formula I compound. Included in the treatment of diabetes is the prevention or attenuation of long term complications such as neuropathy, nephropathy, retinopathy or cataracts.
Yet another aspect of this invention is directed to a method for treating atherosclerosis in a mammal by administering to a mammal suffering from atherosclerosis an atherosclerosis treating amount of a Formula I compound.
Yet another aspect of this invention is directed to a method for treating hyperinsulinemia in a mammal by administering to a mammal suffering from hyperinsulinemia a hyperinsulinemia treating amount of a Formula I compound.
Yet another aspect of this invention is directed to a method for treating hypertension in a mammal by administering to a mammal suffering from hypertension a hypertension treating amount of a Formula I compound.
Yet another aspect of this invention is directed to a method for treating hyperlipidemia in a mammal by administering to a mammal suffering from hyperlipidemia a hyperlipidemia treating amount of a Formula I compound.
Yet another aspect of this invention is directed to a method for preventing a myocardial ischemic injury in a mammal by administering to a mammal at risk for perioperative myocardial ischemic injury a perioperative myocardial ischemic injury preventing amount of a Formula I compound.
Yet another aspect of this invention is directed to a method for preventing a myocardial ischemic injury in a mammal by administering to a mammal at risk for perioperative myocardial ischemic injury a perioperative myocardial ischemic injury preventing amount of a glycogen phosphorylase inhibitor.
This invention is also directed to pharmaceutical compositions which comprise a therapeutically effective amount of a compound of Formula I and a pharmaceutically acceptable carrier.
Preferred compositions include pharmaceutical compositions for the treatment of glycogen phosphorylase dependent diseases or conditions in mammals which comprise a glycogen phosphorylase dependent disease or condition treating amount of a compound of Formula I and a pharmaceutically acceptable carrier.
Another aspect of this invention is directed to pharmaceutical compositions for the treatment of diabetes which comprise a therapeutically effective amount of a glycogen phosphorylase inhibitor;
one or more antidiabetic agents such as insulin and insulin analogs (e.g. LysPro insulin); GLP-1 (7-37) (insulinotropin) and GLP-1 (7-36)-NH2; Sulfonylureas and Analogs: chlorpropamide, glibenclamide, tolbutamide, tolazamide, acetohexamide, glypizide(copyright), glimepiride, repaglinide, meglitinide; Biguanides: metformin, phenformin, buformin; xcex12-Antagonists and Imidazolines: midaglizole, isaglidole, deriglidole, idazoxan, efaroxan, fluparoxan; Other insulin secretagogues: linogliride, A-4166; Glitazones: ciglitazone, pioglitazone, englitazone, troglitazone, darglitazone, BRL49653; Fatty Acid Oxidation Inhibitors: clomoxir, etomoxir; xcex1-Glucosidase inhibitors: acarbose, miglitol, emiglitate, voglibose, MDL-25,637, camiglibose, MDL-73,945; xcex2-Agonists: BRL 35135, BRL 37344, Ro 16-8714, ICI D7114, CL 316,243; Phosphodiesterase Inhibitors: L-386,398; Lipid-lowering Agents: benfluorex; Antiobesity Agents: fenfluramine; Vanadate and vanadium complexes (e.g. naglivan(copyright)) and peroxovanadium complexes; Amylin Antagonists; Glucagon Antagonists; Gluconeogenesis Inhibitors; Somatostatin Analogs; Antilipolytic Agents: nicotinic acid, acipimox, WAG 994; and
optionally a pharmaceutically acceptable carrier.
Preferred pharmaceutical compositions within the immediately preceding group are those compositions wherein the glycogen phosphorylase inhibitor is a compound of Formula I.
Another aspect of this invention is a method of treating diabetes in a mammal with the above described combination compositions.
Glycogen phosphorylase dependent diseases or conditions refers to disorders which are mediated, initiated or maintained, in whole or in part, by the cleavage of the glycogen macromolecule by glycogen phosphorylase enzymes to release glucose-1-phosphate and a new shortened glycogen molecule, These disorders are ameliorated by reduction of or characterized by an elevation of glycogen phosphorylase activity. Examples include diabetes, hyperglycemia, hypercholesterolemia, hypertension, hyperinsulinemia, hyperlipidemia, atherosclerosis and myocardial ischemia.
The term glycogen phosphorylase inhibitor refers to any substance or agent or any combination of substances and/or agents which reduces, retards, or eliminates the enzymatic action of glycogen phosphorylase. The currently known enzymatic action of glycogen phosphorylase is the degradation of glycogen by catalysis of the reversible reaction of a glycogen macromolecule and inorganic phosphate to glucose-1-phosphate and a glycogen macromolecule which is one glucosyl residue shorter than the original glycogen macromolecule (forward direction of glycogenolysis).
The term xe2x80x9ctreatingxe2x80x9d as used herein includes preventative (e.g., prophylactic) and palliative treatment.
By halo is meant chloro, bromo, iodo, or fluoro.
By alkyl is meant straight chain or branched saturated hydrocarbon. Exemplary of such alkyl groups (assuming the designated length encompasses the particular example) are methyl, ethyl, propyl, isopropyl, butyl, sec-butyl, tertiary butyl, pentyl, isopentyl, hexyl and isohexyl.
By alkoxy is meant straight chain or branched saturated alkyl bonded through an oxy. Exemplary of such alkoxy groups (assuming the designated length encompasses the particular example) are methoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, tertiary butoxy, pentoxy, isopentoxy, hexoxy and isohexoxy.
The expression xe2x80x9cpharmaceutically-acceptable anionic saltxe2x80x9d refers to nontoxic anionic salts containing anions such as (but not limited to) chloride, bromide, iodide, sulfate, bisulfate, phosphate, acetate, maleate, fumarate, oxalate, lactate, tartrate, citrate, gluconate, methanesulfonate and 4-toluene-sulfonate.
The expression xe2x80x9cpharmaceutically-acceptable cationic saltxe2x80x9d refers to nontoxic cationic salts such as (but not limited to) sodium, potassium, calcium, magnesium, ammonium or protonated benzathine (N,Nxe2x80x2-dibenzylethylenediamine), choline, ethanolamine, diethanolamine, ethylenediamine, meglamine (N-methyl-glucamine), benethamine (N-benzylphenethylamine), piperazine or tromethamine (2-amino-2-hydroxymethyl-1,3-propanediol).
The expression xe2x80x9cprodrugxe2x80x9d refers to compounds that are drug precursors, which following administration, release the drug in vivo via some chemical or physiological process (e.g., a prodrug on being brought to the physiological pH is converted to the desired drug form). Exemplary prodrugs upon cleavage release the corresponding free acid, and such hydrolyzable ester-forming residues of the compounds of this invention include but are not limited to carboxylic acid substituents (e.g., R6 is carboxy, or R8, R9 or R12 contains carboxy) wherein the free hydrogen is replaced by (C1-C4)alkyl, (C2-C12)alkanoyloxymethyl, 1-(alkanoyloxy)ethyl having from 4 to 9 carbon atoms, 1-methyl-1-(alkanoyloxy)-ethyl having from 5 to 10 carbon atoms, alkoxycarbonyloxymethyl having from 3 to 8 carbon atoms, 1-(alkoxycarbonyloxy)ethyl having from 4 to 7 carbon atoms, 1-methyl-1-(alkoxycarbonyloxy)ethyl having from 5 to 8 carbon atoms, N-(alkoxycarbonyl)aminomethyl having from 3 to 9 carbon atoms, 1-(N-(alkoxycarbonyl)amino)ethyl having from 4 to 10 carbon atoms, 3-phthalidyl, 4-crotonolactonyl, gamma-butyrolacton4-yl, di-N,N-(C1-C2)alkylamino(C2-C3)alkyl (such as xcex2-dimethylaminoethyl), carbamoyl-(C1-C2)alkyl, N,N-di(C1-C2)alkylcarbamoyl-(C1-C2)alkyl and piperidino-, pyrrolidino- or morpholino(C2-C3)alkyl.
Other exemplary prodrugs release an alcohol of Formula I wherein the free hydrogen of the hydroxy substituent (e.g., R5 is hydroxy) is replaced by (C1-C6)alkanoyloxymethyl, 1-((C1-C6)alkanoyloxy)ethyl, 1-methyl-1-((C1-C6)alkanoyloxy)ethyl, (C1-C6)alkoxycarbonyloxymethyl, N-(C1-C6)alkoxycarbonylaminomethyl, succinoyl, (C1-C6)alkanoyl, xcex1-amino(C1-C4)alkanoyl, arylactyl and xcex1-aminoacyl, or xcex1-aminoacyl-xcex1-aminoacyl wherein said xcex1-aminoacyl moieties are independently any of the naturally occurring L-amino acids found in proteins, P(O)(OH)2, xe2x80x94P(O)(O(C1-C6)alkyl)2 or glycosyl (the radical resulting from detachment of the hydroxyl of the hemiacetal of a carbohydrate).
Other exemplary prodrugs include but are not limited to derivatives of Formula I wherein R2 is a free hydrogen which is replaced by R-carbonyl, RO-carbonyl, NRRxe2x80x2-carbonyl where R and Rxe2x80x2 are each independently ((C1-C10)alkyl, (C3-C7)cycloalkyl, benzyl, or R-carbonyl is a natural xcex1-aminoacyl or natural xcex1-aminoacyl-natural xcex1-aminoacyl, xe2x80x94C(OH)C(O)OY wherein (Y is H, (C1-C6)alkyl or benzyl), xe2x80x94C(OY0)Y1 wherein Y0 is (C1-C4) alkyl and Y1 is ((C1-C6)alkyl, carboxy(C1-C6)alkyl, amino(C1-C4)alkyl or mono-N- or di-N,N-(C1-C6)alkylaminoalkyl, xe2x80x94C(Y2)Y3 wherein Y2 is H or methyl and Y3 is mono-N- or di-N,N-(C1-C6)alkylamino, morpholino, piperidin-1-yl or pyrrolidin-1-yl.
Other exemplary prodrugs include but are not limited to derivatives of formula I bearing a hydrolyzable moiety at R3, which release a compound of formula I wherein R3 is a free hydrogen on hydrolysis. Such hydrolyzable moieties at R3 are/include 1-hydroxy(C1-C6)alkyl or 1-hydroxy-1-phenylmethyl.
Other exemplary prodrugs include cyclic structures such as compounds of Formula I wherein R2 and R3 are a common carbon, thus forming a five-membered ring. The linking carbon may be mono- or di-substituted independently with H, (C1-C6)alkyl, (C3-C6)cycloalkyl or phenyl. Alternatively, R3 and R5 may be taken together to form an oxazolidine ring and the number 2 carbon of the oxazolidine ring may be mono- or di-substituted independently with H, (C1-C6)alkyl, (C3-C6)cycloalkyl or phenyl. Alternatively, a prodrug of a Formula I compound includes compounds wherein R5 is taken together with R8 or R9 to form an oxazolidin-4-one ring and the number 2 carbon of said ring may be mono- or di-substituted independently with H, (C1-C6)alkyl, (C3-C6)cycloalkyl, phenyl or oxo.
As used herein, the expressions xe2x80x9creaction-inert solventxe2x80x9d and xe2x80x9cinert solventxe2x80x9d refers to a solvent which does not interact with starting materials, reagents, intermediates or products in a manner which adversely affects the yield of the desired product.
The chemist of ordinary skill will recognize that certain compounds of this invention will contain one or more atoms which may be in a particular stereochemical or geometric configuration, giving rise to stereoisomers and configurational isomers. All such isomers and mixtures thereof are included in this invention. Hydrates of the compounds of this invention are also included.
The chemist of ordinary skill will recognize that certain combinations of heteroatom-containing substituents listed in this invention define compounds which will be less stable under physiological conditions (e.g. those containing acetal or aminal linkages). Accordingly, such compounds are less preferred.
The term xe2x80x9cRx ringxe2x80x9d wherein x is an integer, for example xe2x80x9cR9 ringxe2x80x9d, xe2x80x9cR12 ringxe2x80x9d or xe2x80x9cR4ringxe2x80x9d as used herein in reference to substitution on the ring refers to moieties wherein the ring is Rx and also wherein the ring is contained within Rx.
As used herein the term mono-N- or di-N,N-(C1-Cx)alkyl . . . refers to the (C1-Cx) alkyl moiety taken independently when it is di-N,N-(C1-Cx)alkyl . . . ; (x refers to an integer).
Other features and advantages will be apparent from the specification and claims which describe the invention.
In general the compounds of Formula I can be made by processes which include processes known in the chemical arts, particularly in light of the description contained herein. Certain processes for the manufacture of Formula I compounds are provided as further features of the invention and are illustrated by the following reaction schemes. 
According to Reaction Scheme I the Formula I compounds, wherein R1, R10, R11, A, R2, R3, R4, R5, R6 and R7 are as defined above may be prepared by either of two general processes. In the first process the desired Formula I compound may be prepared by coupling the appropriate Formula I indole-2-carboxylic acid or indoline-2-carboxylic acid with the appropriate Formula III amine (i.e., acylating the amine). In the second process the desired Formula I compound may be prepared by coupling the appropriate Formula IV compound (i.e., a Formula I compound wherein R6 is carboxy) with the appropriate alcohol or formula R8R9NH or R12H amine or alcohol, wherein R8, R9 and R12 are as defined above (i.e., acylating the amine or alcohol).
Typically, the Formula II compound is combined with the Formula III compound (or Formula IV compound is combined with the appropriate amine (e.g., R12H or R8R9NH)) or alcohol in the presence of a suitable coupling agent. A suitable coupling agent is one which transforms a carboxylic acid into a reactive species which forms an amide or ester linkage on reaction with an amine or alcohol, respectively.
The coupling agent may be a reagent which effects this condensation in a one pot process when mixed together with the carboxylic acid and amine or alcohol. If the acid is to be condensed with an alcohol it is preferable to employ a large excess of the alcohol as the reaction solvent, with or without 1.0 to 1.5 equivalent added dimethylaminopyridine. Exemplary coupling reagents are 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride-hydroxybenzotriazole (DEC/HBT), carbonylduimidazole, dicyclohexylcarbodiimide/hydroxybenzotriazole (HBT), 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline (EEDQ), carbonyldiimidazole/HBT, and diethylphosphorylcyanide. The coupling is performed in an inert solvent, preferably an aprotic solvent at a temperature of about xe2x88x9220xc2x0 C. to about 50xc2x0 C. for about 1 to about 48 hours. Exemplary solvents include acetonitrile, dichloromethane, dimethylformamide and chloroform. An example of a suitable coupling procedure is Procedure A, contained herein (just prior to the EXAMPLES).
The coupling agent may also be that agent which converts the carboxylic acid to an activated intermediate which is isolated and/or formed in a first step and allowed to react with the amine or alcohol in a second step. Examples of such coupling agents and activated intermediates are thionyl chloride or oxalyl chloride to form the acid chloride, cyanuric fluoride to form an acid fluoride or an alkyl chloroformate such as isobutyl or isopropenyl chloroformate (with a tertiary amine base) to form a mixed anhydride of the carboxylic acid. If the coupling agent is oxalyl chloride it is advantageous to employ a small amount of dimethylformamide as cosolvent with another solvent (such as dichloromethane) to catalyze the formation of the acid chloride. Use of these coupling agents and appropriate selection of solvents and temperatures are known to those skilled in the art or can be readily determined from the literature. These and other exemplary conditions useful for coupling carboxylic acids are described in Houben-Weyl, Vol XV, part II, E. Wunsch, Ed., G. Theime Verlag, 1974, Stuttgart, and M. Bodansky, Principles of Peptide Synthesis, Springer-Verlag Berlin 1984, and The Peptides. Analysis, Synthesis and Biology (ed. E. Gross and J. Meienhofer), vols 1-5 (Academic Press NY 1979-1983).
The Formula IV compounds wherein R1, R10, R11, A, R2, R3, R4, R6, and R7 are as defined above may be prepared from the corresponding Formula V ester (i.e., Formula I compounds wherein R6 is (C1-C5)alkoxycarbonyl or benzyloxycarbonyl) by hydrolysis with aqueous alkali at a temperature of about xe2x88x9220xc2x0 C. to about 100xc2x0 C., typically at about 20xc2x0 C., for about 30 minutes to about 24 hours.
Alternatively, Formula IV compounds are prepared by activation of a Formula II indole carboxylic acid with a coupling agent (as described above) which gives an activated intermediate (such as an acid chloride, acid fluoride, or mixed anhydride) which is then allowed to react with a compound of Formula III wherein R3, R4, R5, and R7 are as described above and R5 is carboxy, in a suitable solvent in the presence of a suitable base. Suitable solvents include wateror methanol or a mixture thereof, together with a cosolvent such as dichloromethane, tetrahydrofuran, or dioxane. Suitable bases include sodium, potassium or lithium hydroxides, sodium or potassium bicarbonate, sodium or potassium carbonate, or potassium carbonate together with tetrabutyl ammonium bromide (1 equivalent) in sufficient quantity to consume the acid liberated in the reaction (generally that quantity sufficient to maintain the pH of the reaction at greater than 8). The base may be added incrementally together with the activated intermediate to effect proper pH control of the reaction. The reaction is conducted generally between xe2x88x9220xc2x0 C. and 50xc2x0 C. Isolation procedures are tailored by one skilled in the art to remove impurities, but typically consist of removal of water-miscible cosolvents by evaporation, extraction of impurities at high pH with an organic solvent, acidification to low pH (1-2) and filtration or extraction of the desired product with a suitable solvent such as ethyl acetate or dichloromethane.
The Formula V compound may be prepared by coupling the appropriate Formula III compound wherein R6 is alkoxycarbonyl and the appropriate Formula II compound in an analogous procedure to that described above (e.g., Procedure A).
Alternatively, Formula I compounds which contain sulfur atoms in the sulfoxide or sulfone oxidation state may be prepared from the corresponding Formula I compounds having the sulfur atom in the unoxidized form, by treatment with a suitable oxidizing agent, such as with m-chloroperoxybenzoic acid in dichloromethane at a temperature of about 0xc2x0 C. to about 25xc2x0 C. for about 1 to about 48 hours using about 1 to about 1.3 equivalent for conversion to the sulfoxide oxidation state and greater than about 2 equivalents for conversion to the sulfone oxidation state.
Alternatively, the Formula I compounds that are mono- or di-alkylated on R5 aminoalkoxy may be prepared from the corresponding Formula I compound wherein R5 is aminoalkoxy by monoalkylation or dialkylation on the R5 amine to prepare the desired Formula I compound. Such a mono- or di-alkylation may be conducted by treatment of the R5 aminoalkoxy compound with 1 equivalent of the appropriate carbonyl compound (for monoalkylation) or greater than 2 equivalents of the appropriate carbonyl compound (for dialkylation) and a suitable reducing agent in a suitable solvent. Suitable reducing conditions include sodium cyanoborohydride or sodium borohydride in methanol or ethanol, or hydrogen/hydrogenation catalyst (such as palladium on carbon) in a polar solvent such as water, methanol, or ethanol at about 0xc2x0 C. to 60xc2x0 C. for 1 to 48 hours.
Alternatively, the Formula I compounds, wherein R5 is alkanoyloxy (RCOOxe2x80x94), are prepared by O-acylation of the appropriate Formula I compound with an appropriate acid chloride or other activated acid derivative in the presence, if necessary, of a suitable base, (e.g., tertiary amine base such as trialkylamine or pyridine), preferably in an aprotic solvent such as tetrahydrofuran or dichloromethane, at a temperature of about 0xc2x0 C. to about 50xc2x0 C., for about 0.5 to about 48 hours.
Alternatively, the Formula I compounds wherein R5 and R7 are taken together to be oxo are prepared by oxidizing a corresponding Formula I compound, for example, wherein R5 is hydroxy and R7 is H, with a suitable oxidizing agent. Exemplary oxidizing agents include the Dess-Martin reagent in dichloromethane, a carbodiimide and dimethylsulfoxide and acid catalyst (Pfitzner-Moffatt conditions or modifications thereof, such as employing a water-soluble carbodiimide) or Swern-type reactions (e.g., oxalyl chloride/DMSO/triethylamine). The Formula I compounds having other oxidation sensitive functionality may benefit from appropriate protection and deprotection of such functionality.
Some of the preparation methods described herein may require protection of remote functionality (i.e., primary amine, secondary amine, carboxyl in Formula I precursors). The need for such protection will vary depending on the nature of the remote functionality and the conditions of the preparation methods. The need for such protection is readily determined by one skilled in the art. The use of such protection/deprotection methods is also within the skill in the art. For a general description of protecting groups and their use, see T. W. Greene, Protective Groups in Organic Synthesis, John Wiley and Sons, New York, 1991.
For example, in Reaction Scheme I certain Formula I compounds contain primary amine, secondary amine or carboxylic acid functionality in the part of the molecule defined by R5 or R6 which may interfere with the intended coupling reaction of Reaction Scheme I if the Formula III intermediate, or R12H or R8R9NH amine is left unprotected. Accordingly, the primary or secondary amine functionality may be protected, where it is present in the R5 or R6 moieties of the Formula III intermediate or amine (R8R9NH or R12H) by an appropriate protecting group during the coupling reaction of Reaction Scheme I. The product of such coupling reaction is a Formula I compound containing the protecting group. This protecting group is removed in a subsequent step to provide the Formula I compound. Suitable protecting groups for amine and carboxylic acid protection include those protecting groups commonly used in peptide synthesis (such as N-t-butoxycarbonyl, N-carbobenzyloxy, and 9-fluorenylmethylenoxycarbonyl for amines and lower alkyl or benzyl esters for carboxylic acids) which are not chemically reactive under the coupling conditions described above (and immediately preceding the Examples herein as Procedure A) and can be removed without chemically altering other functionality in the Formula I compound.
The starting indole-2-carboxylic acids and indoline-2-carboxylic acids used in Reaction Scheme I, when not commercially available or known in the prior art (such art is extensively published), are available by conventional synthetic methods. For example, according to Reaction Scheme II the Formula VII indole ester may be prepared from the Formula VI compound (wherein Q is selected to achieve the desired A as defined above) via a Fischer Indole synthesis (see The Fischer Indole Synthesis Robinson, B. (Wiley, New York, 1982)) followed by saponification of the resulting Formula VII indole ester to yield the corresponding Formula VIII acid. The starting aryl hydrazone may be prepared by condensation of a readily available hydrazine with the appropriate carbonyl derivative or via the Japp-Kiingeman reaction (see Organic Reactions, Phillips, R. R., 1959, 10, 143).
Alternatively, the Formula VIIIA indole 2-carboxylic acid may be prepared by condensation of a Formula IX ortho methyl nitro compound with an oxalate ester to yield the Formula X indole ester followed by reduction of the nitro group and subsequent hydrolysis.
This three step process is known as the Reissert indole synthesis (Reissert, Chemische Berichte 1897, 30, 1030). Conditions for accomplishing this sequence, and references thereto, are described in the literature (Kermack, et al., J. Chem. Soc. 1921, 119, 1602; Cannon et al., J. Med. Chem. 1981, 24, 238; Julian, et al in Heterocyclic Compounds, vol 3 (Wiley, New York, N.Y., 1962, R. C. Elderfield, ed.) p 18). An example of the specific implementation of this sequence is Examples 10A-10C herein.
3-Halo-5-chloro-1H-indole-2-carboxylic acids may also be prepared by halogenation of 5-chloro-1H-indole-2-carboxylic acids.
Alternatively, (to Reaction Scheme II) the Formula XIV substituted indolines may be prepared by reduction of the corresponding Formula XV indoles with a reducing agent such as magnesium in methanol at a temperature of about 25xc2x0 C. to about 65xc2x0 C. for about 1 to about 48 hours (Reaction Scheme III).
Formula XVI indoline carboxylic acids are prepared by saponification of the corresponding Formula XVII ester (Reaction Scheme III). The Formula XVII compound is prepared by reduction of the corresponding Formula VII indole ester with a reducing agent such as magnesium in methanol as described for the conversion of the Formula XV compound to the Formula XIV compound above.
The following paragraphs describe how to prepare the various amines which are used in the above Reaction Schemes.
According to Reaction Scheme IV the Formula XXII compounds (the Formula III amines of Reaction Scheme I wherein R5 is OH, R7 is H and R6 is an ester) or Formula XXVI compounds (R6 is C(O)NR8R9 or C(O)R12) are prepared starting from a Formula XX N-protected (denoted by PT) aldehyde. The Formula XX aldehyde or the sodium bisulfite adduct of a Formula XX aldehyde is treated with potassium or sodium cyanide in aqueous solution with a cosolvent such as dioxane or ethyl acetate at a temperature of about 0xc2x0 C. to about 50xc2x0 C. to provide a Formula XXI cyanohydrin. The Formula XXI cyanohydrin is treated with an alcohol (e.g., (C1-C6)alkanol such as methanol) and a strong acid catalyst such as hydrogen chloride at a temperature of about 0xc2x0 C. to about 50xc2x0 C., followed by addition of water, if necessary. The protecting group (PT) is then removed, if still present, by an appropriate deprotection method yielding a Formula XXII compound. For example, if the Formula XX N-protecting group PT is tert-butoxycarbonyl (t-Boc), the Formula XXIII compound is directly formed from the Formula XXI compound, and addition of water is not necessary. The Formula XXII compound may be protected on nitrogen with an appropriate protecting group to form a Formula XXIII compound followed by hydrolysis of the ester with aqueous alkali at a temperature of about 0xc2x0 C. to about 50xc2x0 C. in a reaction-inert solvent resulting in the corresponding Formula XXIV hydroxy acid. The Formula XXIV compound is coupled (in an analogous procedure to the coupling process described in Reaction Scheme I) with an appropriate R8R9NH or HR12 amine to form a Formula XXV compound, which is then deprotected resulting in the Formula XXVI compound (i.e., Formula III compound wherein R5 is OH, R7 is H and R6 is C(O)R12 or C(O)NR8R9. An example of the conversion of a Formula XXI cyanohydrin to the corresponding Formula XXII methyl ester with removal of the t-boc protecting group is provided in PCT publication WO/9325574, Example 1a. Other examples wherein a cyanohydrin is converted to Formula XXIII lower alkyl esters may be found in U.S. Pat. No. 4,814,342, and EPO publication O438233.
Certain Formula I compounds are stereoisomeric by virtue of the stereochemical configuration at the carbons labeled a and b. One skilled in the art may prepare Formula XXII and XXVI intermediates with the desired stereochemistry according to Reaction Scheme IV. For example, the Formula XX aldehyde is available in either enantiomeric form (stereochemistry at a) by literature procedures outlined below (see Reaction Scheme V). The Formula XXI cyanohydrin may be prepared from the Formula XX compound by treatment with sodium or potassium cyanide as described above while maintaining the stereochemistry at carbon a resulting in a mixture of stereoisomers at carbon b.
The skilled chemist may employ crystallization at this stage to separate isomers or purify one isomer.
For example, the preparation of the Formula XXI compound wherein PT is Boc, R3 is H, R4 is benzyl and the stereochemistry of carbons a and b is (S) and (R) respectively, employing this route together with purification by recrystallization is described in Biochemistry 1992, 31, 8125-8141.
Alternatively, isomer separation may be effected by chromatography or recrystallization techniques after conversion of a compound of formula XXI (mixture of isomers) to a compound of formula XXII, XXIII, XXIV, XXV, XXVI, V, IV, or I by the procedures and/or sequences described herein. Formula XXI intermediates of a specific stereochemistry at carbons a and b are converted to Formula XXII intermediates with retention of this stereochemistry by treatment with an alcohol and a strong acid catalyst, followed by addition of water, if necessary, as described above.
Alternatively, the desired isomer of the Formula XXI compound may also be obtained by derivatization of the Formula XXI intermediate and chromatographic separation of the diastereomeric derivatives (for example with trimethylsilyl chloride (TMS) or t-butyldimethylsilyl chloride TBDMS) to give O-TMS or O-TBDMS derivatives). For example, Example 24D (contained herein) describes the separation of Formula XXI diastereomeric derivatives. A silyl derivative of a Formula XXI intermediate having a single stereoisomeric form at carbons a and b is converted with retention of stereochemistry to a Formula XXII intermediate (if the silyl group is not removed in this step it is removed subsequently by an appropriate method, such as treatment with tetrabutylammonium fluoride in tetrahydrofuran), by the method described above for the conversion of the Formula XXI compound to the Formula XXII compound (see Example 24C contained herein for conversion of a silyl derivative of Formula XXI compound to a single isomer of Formula XXII with loss of the silyl group).
According to Reaction Scheme V the Formula XX aldehydes (starting materials for Reaction Scheme IV) are prepared from the corresponding Formula XXX amino acids. The Formula XXX amino acid is protected on nitrogen with a protecting group (PT) (such as Boc). The protected compound is esterified with an alcohol and converted to an ester, preferably the methyl or ethyl ester of the Formula XXXI compound. This may be accomplished by treating the Formula XXX compound with methyl or ethyl iodide in the presence of a suitable base (e.g., K2CO3) in a polar solvent such as dimethylformamide. The Formula XXXI compound is reduced, for example, with diisobutylaluminum hydride in hexane or toluene, or a mixture thereof, at a temperature of about xe2x88x9278xc2x0 C. to about xe2x88x9250xc2x0 C. followed by quenching with methanol at xe2x88x9278xc2x0 C. as described in J. Med. Chem., 1985, 28, 1779-1790 to form the Formula XX aldehyde. Alternatively (not depicted in Reaction Scheme V), analogous N-methoxymethylamides corresponding to the Formula XXXI compound, wherein the alcohol substituent of the ester is replaced by N(OMe)Me, are formed from a Formula XXX compound, N,O-dimethylhydroxylamine and a suitable coupling agent (e.g., 1-(3-dimethylaminopropyl)3-ethylcarbodiimide hydrochloride (DEC) as in Procedure A. The resulting compound is reduced, for example, with lithium aluminum hydride in a reaction-inert solvent such as ether or tetrahydrofuran at a temperature of about 0xc2x0 C. to about 25xc2x0 C. to form the Formula XX aldehyde. This two-step method is general for the conversion of N-protected xcex1-amino acids to Formula XX aldehydes (Fehrentz and Castro, Synthesis 1983, 676-678).
Alternatively Formula XX aldehydes may be prepared by oxidation of Formula XXXIII protected aminoalcohols, for example, with pyridine-SO3 at a temperature of about xe2x88x9210xc2x0 C. to about 40xc2x0 C. in a reaction-inert solvent, preferably dimethylsulfoxide. Formula XXXIII protected aminoalcohols, if not commercially available, may be prepared by protection of Formula XXXII aminoalcohols. The Formula XXXII aminoalcohols are prepared by reduction of Formula XXX amino acids. This reduction is accomplished by treatment of Formula XXX compounds with lithium aluminum hydride according to the procedure described by Dickman et al., Organic Syntheses; Wiley: New York, 1990; Collect. Vol. VII, p 530, or with sulfuric acid-sodium borohydride by the procedure of Abiko and Masamune, Tetrahedron Lett. 1992 333, 5517-5518, or with sodium borohydride-iodine according to the procedure of McKennon and Meyers, J. Org. Chem. 1993, 58, 3568-3571, who also reviewed other suitable procedures for converting Formula XXX amino acids to Formula XXXII amino alcohols.
According to Reaction Scheme VI the Formula XXX compounds utilized in Reaction Scheme V may be prepared as follows. The Formula XLI amino acids may be prepared by N-alkylation of the Formula XL protected (PT) amino acids by treatment with an appropriate base and alkylating agent. Specific procedures for this alkylation are described by Benoiton, Can. J. Chem 1977, 55, 906-910, and Hansen, J. Org. Chem. 1985, 50 945-950. For example, when R3 is methyl, sodium hydride and methyl iodide in tetrahydrofuran are utilized. Deprotection of the Formula XLI compound yields the desired Formula XXX compound.
Alternatively, a Formula XLII amino acid may be N-alkylated by a three-step sequence involving reductive benzylation (such as with benzaldehyde, Pd/C-catalyzed hydrogenation) to give the mono-N-benzyl derivative and reductive amination with the appropriate acyl compound (for example with formaldehyde and sodium cyanoborohydride to introduce R3 as methyl) to give the N-Benzyl, Nxe2x80x94R3-substituted amino acid. The N-benzyl protecting group is conveniently removed (for example by hydrogenation with an appropriate catalyst) to yield the Formula XXX compound. Specific conditions for this three step alkylation procedure are described by Reinhold et at., J. Med. Chem., 1968, 11, 258-260.
The immediately preceding preparation may also be used to introduce an R3 moiety into the Formula XLIV intermediate to form the Formula XLV intermediate (which is a Formula III intermediate wherein R7 is OH). The immediately preceding preparation may also be used to introduce an R3 moiety into a Formula IIIa intermediate (which is a Formula III intermediate wherein R3 is H).
The amino acids used in the schemes herein (e.g., XL, XLII), if not commercially available, or reported in the literature, may be prepared by a variety of methods known to those skilled in the art. For example, the Strecker synthesis or variations thereof may be used. Accordingly, an aldehyde (R4CHO), sodium or potassium cyanide and ammonium chloride react to form the corresponding aminonitrile. The aminonitrile is hydrolyzed with mineral acid to form the desired Formula XLII R4C(NH2)COOH amino acid. Alternatively, the Bucherer-Berg method may be used wherein a hydantoin is formed by heating an aldehyde (R4CHO) with ammonium carbonate and potassium cyanide followed by hydrolysis (for example, with barium hydroxide in refluxing dioxane) with acid or base to form the desired Formula XLII R4C(NH2)COOH amino acid.
Other methods for synthesis of xcex1-amino acids are also reported in the literature which would permit one skilled in the art to prepare the desired Formula XLII R4C(NH2)COOH intermediate necessary for the synthesis of Formula I compounds.
Suitable methods for the synthesis or resolution of Formula XLII compounds are found in reviews by Duthaler (Tetrahedron 1994, 50, 1539-1650), or by Williams (R. M. Williams, Synthesis of optically active amino acids. Pergamon: Oxford, U.K., 1989).
A specific method for the synthesis of a Formula XLII intermediate in either enantiomeric form from the corresponding R4X (X=Cl, Br, or I) intermediate is the procedure of Pirrung and Krishnamurthy (J. Org. Chem. 1993, 58, 957-958), or by the procedure of O""Donnell, et al. (J. Am. Chem. Soc. 1989, 111, 2353-2355). The required R4X intermediates are readily prepared by many methods familiar to the chemist skilled in the art. For example, those compounds when R4X is ArCH2X may be prepared by radical halogenation of the compound ArCH3 or by formylation of the arene Arxe2x80x94H and conversion of the alcohol to the bromide.
Another specific method for the synthesis of Formula XLII intermediates in either enantiomeric form is that of Corey and Link (J. Am. Chem. Soc. 1992, 114, 1906-1908). Thus, an intermediate of formula R4COCCl3 is reduced enantiospecifically to intermediate R4CH(OH)CCl3, which is converted on treatment with azide and base to an intermediate R4CH(N3)COOH, which is reduced by catalytic hydrogenation to the desired Formula XLII compound. The requisite trichloromethyl ketone R4COCCl3 is obtained by reaction of the aldehyde R4CHO with trichloromethide anion followed by oxidation (Gallina and Giordano, Synthesis 1989, 466-468).
Formula III intermediate amines (used in Reaction Scheme I), wherein R5 and R7 are H may be prepared according to Reaction Scheme VII. A Formula L amino acid (suitably protected (PT) is activated by conversion to the acid chloride, fluoride or mixed anhydride (e.g., with isobutyl chloroformate and triethylamine in an inert solvent such as tetrahydrofuran or dioxane at about xe2x88x920xc2x0 C. to about xe2x88x9240xc2x0 C.) and the activated intermediate treated with diazomethane to give the Formula LI diazoketone. The Formula LI diazoketone is treated with an alcohol (ROH) (e.g., (C1-C6)alkanol such as methanol), and a suitable catalyst such as heat, silver oxide or silver benzoate to prepare the Formula LII ester. The Formula LII ester is deprotected to form the Formula IIIA compound (via Wolff rearrangement). Alternatively the Formula LII ester is hydrolyzed, with for example alkali, and coupled with the appropriate R12H or HNR8R9 amine to prepare the Formula IIIB compound as described previously.
According to Reaction Scheme VIII the Formula III intermediate amines wherein R5 is an oxygen linked substituent (e.g., alkoxy) (used in Reaction Scheme I) may be prepared as follows. The Formula LXI compound is alkylated on oxygen by treatment with an appropriate alkylating agent (e.g., alkyliodide, alkylbromide, alkylchloride or alkyltosylate) and sufficient base to form the alkoxide (sodium or potassium hydride) in a-suitable polar aprotic solvent (e.g., dimethylformamide or tetrahydrofuran) at a temperature of about 0xc2x0 C. to about 150xc2x0 C. resulting in a formula LXII compound. The Formula LXII compound is deprotected to afford the desired amine intermediate.
The Formula III intermediate amines wherein R5 is (C1-C6) alkoxycarbonylalkoxy (used in Reaction Scheme I) may be prepared as follows. The Formula LXI compound is alkylated with a halo-alkanoate ester to form a Formula LXIII compound which is then deprotected to form the desired amine. The corresponding acid may be prepared by hydrolysis of the ester using aqueous alkali in an appropriate solvent. Those Formula III amines wherein R6 contains an ester and R5 contains a carboxy may be prepared from the Formula LXIII amine (as prepared above in this paragraph), wherein R5 contains the carboxylic acid functionality protected as the t-butyl ester by treatment with anhydrous acid to provide the corresponding acid at R5 without hydrolyzing the ester at the R6 position.
The Formula LXVI compounds (Formula III intermediate amines wherein R6 is protected aminoalkoxy) may be prepared from the Formula LXI compound. The Formula LXI compound is alkylated with a halo-alkane-nitrile to form the Formula LXIV compound. The Formula LXIV compound is reduced to the primary amine by treatment with hydrogen and an appropriate catalyst (e.g., rhodium-on-carbon) in the pressence of ammonia in preferably a polar, protic solvent such as water, methanol or ethanol to give the Formula LXV primary amine. The Formula LXV compound is protected on nitrogen with a protecting group (PT1), which is orthogonal to the other protecting group (PT), followed by deprotection of the PT protecting group to yield the desired Formula III compound. The protected Formula III compound is coupled with the appropriate Formula II compound and the resulting protected Formula I compound is deprotected.
The Formula LXIII and LXIV compounds wherein n is two are preferably prepared by treatment of the Formula LXI compound with an excess of acrylate ester or acrylonitrile, respectively, in the presence of a suitable base, such as potassium or sodium hydroxide, in a suitable solvent, preferably a polar protic solvent.
According to Reaction Scheme IX the Formula LXVII and Formula LXIX compounds (Formula III compounds wherein R5 is F or R5 and R7 are both F) may be prepared from the Formula LXI compound. The Formula LXI compound is treated with a suitable fluorinating agent such as diethylaminosulfur trifluoride in a reaction-inert solvent such as an aprotic solvent, preferably dichloromethane, to form the Formula LXVII compound. The Formula LXVII compound is conveniently deprotected.
The Formula LXI compound is oxidized to the Formula LXVIII compound utilizing the conditions described above for the preparation of the Formula I compounds wherein R5 and R7 together form oxo. The Formula LXVIII compound is difluorinated under suitable conditions (e.g., diethylaminosulfur trifluoride in dichloromethane).
According to Reaction Scheme X the Formula LXXIII compound or Formula LXIV compound wherein R7 is alkyl (i.e., Formula III compound wherein R7 is alkyl) are prepared from the Formula LXX compound (also see Reaction Scheme V for analogous amine preparation). The Formula LXX compound is treated with an organometallic reagent R7M and the resulting secondary alcohol oxidized as in the directly preceding paragraph to form the Formula LXXI compound. The Formula LXXI compound is converted via the Formula LXXII cyanohydrin to the Formula LXXIII compound using the same conditions that are used to convert the Formula XXI compound to the Formula XXII compound in Reaction Scheme IV.
Alternatively, the Formula LXXII compound is converted to the Formula LXIV compound as described for the conversion of the cyano intermediate to the amide in Reaction Scheme V.
A compound of the formula R8NH2 or R9NH2 is monoalkylated with a carbonyl compound corresponding to R8 or R9, respectively, under appropriate reductive amination conditions, to give a formula R8R9NH amine. To avoid dialkylation, it may be preferable to protect the amines (R8NH2 or R9NH2) with a suitable protecting group PT to give R8(PT)NH or R9(PT)NH, for example by reaction with benzaldehyde and a reducing agent. The protected amines are monoalkylated with a carbonyl compound corresponding to R9 or R8 respectively, under suitable reductive amination conditions, to give R8R9N(PT). The protecting group (PT) is removed (e.g. by exhaustive catalytic hydrogenation when PT is benzyl) to give a compound of formula R8R9NH. Appropriate reductive amination conditions are available from the literature to one skilled in the art. These conditions include those reported by Borch et al. (J. Am. Chem. Soc. 1971, 2897-2904) and those reviewed by Emerson (Organic Reactions, Wiley: New York, 1948 (14), 174), Hutchins et al. (Org. Prep. Proced. Int 1979 (11), 20, and Lane et al. (Synthesis 1975, 135). Reductive amination conditions favoring N-monoalkylation include those reported by Morales, et al. (Synthetic Communications 1984, 1213-1220) and Verardo et al. (Synthesis 1992 121-125). The R8NH2 or R8NH2 amines may also be monoalkylated with R9X or R8X, respectively, where X is chloride, bromide, tosylate or mesylate. Alternatively, an intermediate of formula R8(PT)NH or R9(PT)NH may be alkylated with R9X or R8X, and the protecting group removed to give a compound of formula R8R9NH.
Additional methods may be used to prepare formula R8R9NH amines wherein R8xe2x80x94NH or R9xe2x80x94NH are oxygen-nitrogen linked. Thus a readily available compound of formula (C1-C4)alkoxycarbonyl-NHOH or NH2CONHOH is dialkylated on nitrogen and oxygen by treatment with base and excess suitable alkylating agent (R-X) to give the corresponding (C1-C4)alkoxycarbonyl-N(R)OR which is then hydrolyzed to give a compound of formula R8R9NH (wherein R8=R9=R). Suitable conditions, base, and alkylating agent include those described by Goel and Krolls (Org. Prep. Proced. Int. 1987, 19, 75-78) and Major and Fleck (J. Am. Chem. Soc. 1928, 50, 1479). Alternatively, a formula NH2CONH(OH) amine may be sequentially alkylated, first on oxygen to give NH2CONH(ORxe2x80x2), then on nitrogen to give NH2CON(Rxe2x80x3)(ORxe2x80x2), by successive treatment with the alkylating agents Rxe2x80x2X and Rxe2x80x3X, respectively, in the presence of a suitable base. Suitable base and alkylating agents include those described by Kreutzkamp and Messinger (Chem. Ber. 100, 3463-3465 (1967) and Danen et al (J. Am. Chem. Soc. 1973, 95, 5716-5724). Hydrolysis of these alkylated hydroxyurea derivatives yields the amines Rxe2x80x2ONH2 and Rxe2x80x2ONHRxe2x80x3, which correspond to certain formula R8R9NH amines. The chemist skilled in the art can adapt the procedures described in this paragraph to other alkylating agents R, Rxe2x80x2 and Rxe2x80x3-X to prepare other amines of formula R8R9NH wherein R8xe2x80x94N or R9xe2x80x94N are oxygen-nitrogen linked. Uno et al (SynLett 1991, 559-560) describe the BF3-catalyzed addition of an organometallic reagent R-Li to an O-alkyl oxime of formula Rxe2x80x2CHxe2x95x90Nxe2x80x94ORxe2x80x3, to give compounds of formula Rxe2x80x2RCHxe2x80x94NH(ORxe2x80x3). This route may also be used to give compounds of formula R8R9NH wherein one of R8xe2x80x94NH or R9xe2x80x94NH are oxygen-nitrogen linked.
Prodrugs of this invention where a carboxyl group in a carboxylic acid of Formula I is replaced by an ester may be prepared by combining the carboxylic acid with the appropriate alkyl halide in the presence of a base such as potassium carbonate in an inert solvent such as dimethylformamide at a temperature of about 0xc2x0 C. to 100xc2x0 C. for about 1 to about 24 hours. Alternatively the acid is combined with appropriate alcohol as solvent in the presence of a catalytic amount of acid such as concentrated sulfuric acid at a temperature of about 20xc2x0 C. to 120xc2x0 C., preferably at reflux, for about 1 hour to about 24 hours. Another method is the reaction of the acid with a stoichiometric amount of the alcohol in the presence of a catalytic amout of acid in an inert solvent such as tetrahydrofuran, with concomitant removal of the water being produced by physical (e.g. Dean-Stark trap) or chemical (e.g. molecular sieves) means.
Prodrugs of this invention where an alcohol function has been derivatized as an ether may be prepared by combining the alcohol with the appropriate alkyl bromide or iodide in the presence of a base such as potassium carbonate in an inert solvent such as dimethylformamide at a temperature of about 0xc2x0 C. to 100xc2x0 C. for about 1 to about 24 hours. Alkanoylaminomethyl ethers may be obtained by reaction of the alcohol with a bis-(alkanoylamino)methane in the presence of a catalytic amount of acid in an inert solvent such as tetrahydrofuran, according to a method described in U.S. Pat. No. 4,997,984. Alternatively, these compounds may be prepared by the methods described by Hoffman et al. in J. Org. Chem. 1994, 59, 3530.
The dialkylphosphate esters may be prepared by reaction of the alcohol with a dialkyl chlorophosphate in the presence of a base in an inert solvent such as tetrahydrofuran. The dihydrogen phosphates may be prepared by reaction of the alcohol with a diaryl or dibenzyl chlorophosphate as described above, followed by hydrolysis or hydrogenation in the presence of a noble metal catalyst, respectively.
Glycosides are prepared by reaction of the alcohol and a carbohydrate in an inert solvent such as toluene in the presence of acid. Typically the water formed in the reaction is removed as it is being formed as described above. An alternate procedure is the reaction of the alcohol with a suitably protected glycosyl halide in the presence of base followed by deprotection.
N-(1-hydroxyalkyl) amides, N-(1-hydroxy-1-(alkoxycarbonyl)methyl) amides or compounds where R2 has been replaced by C(OH)C(O)OY may be prepared by the reaction of the parent amide or indole with the appropriate aldehyde under neutral or basic conditions (e.g. sodium ethoxide in ethanol) at temperatures between 25 and 70xc2x0 C. N-alkoxymethyl indoles or N-1-(alkoxy)alkyl indoles can be obtained by reaction of the N-unsubstituted indole with the necessary alkyl halide in the presence of a base in an inert solvent. 1-(N,N-dialkylaminomethyl) indole, 1-(1-(N,N-dialkylamino)ethyl) indole and N,N-dialkylaminomethyl amides (e.g. R3=CH2N(CH3)2) may be prepared by the reaction of the parent Nxe2x80x94H compound with the appropriate aldehyde and amine in an alcoholic solvent at 25 to 70xc2x0 C.
The aforementioned cyclic prodrugs (e.g., the prodrugs of this invention where R2 and R3 are a common carbon) may be prepared by reaction of the parent compound (drug) with an aldehyde or ketone or its dimethyl acetal in an inert solvent in the presence of a catalytic amount of acid with concomitant water or methanol removal. Alternatively, these compounds may be prepared by reaction of the amino alcohol or hydroxy amide with a gem-dibromo alkane in the presence of base (e.g. potassium carbonate) in an inert solvent (e.g. dimethylformamide).
The starting materials and reagents for the above described reaction schemes (e.g., amines, substituted indole carboxylic acids, substituted indoline carboxylic acids, amino acids), although the preparation of most of which are described above, are also readily available or can be easily synthesized by those skilled in the art using conventional methods of organic synthesis. For example, many of the intermediates used herein to prepare compounds of Formula I are, are related to, or are derived from amino acids found in nature, in which there is a large scientific interest and commercial need, and accordingly many such intermediates are commercially available or are reported in the literature or are easily prepared from other commonly available substances by methods which are reported in the literature. Such intermediates include, for example, Formula XX, Formula XXX, Formula XXXI, and Formula XXXII compounds.
The compounds of Formula I have asymmetric carbon atoms and therefore are enantiomers or diastereomers. Diasteromeric mixtures can be separated into their individual diastereomers on the basis of their physical chemical differences by methods known per se., for example, by chromatography and/or fractional crystallization. Enantiomers (e.g., of Formula III, VIII or IX) can be separated by converting the enantiomeric mixture into a diasteromeric mixture by reaction with an appropriate optically active compound (e.g., alcohol), separating the diastereomers and converting (e.g., hydrolyzing) the individual diastereomers to the corresponding pure enantiomers. All such isomers, including diastereomers, enantiomers and mixtures thereof are considered as part of this invention.
Although many compounds of this invention are not ionizable at physiological conditions, some of the compounds of this invention are ionizable at physiological conditions. Thus, for example some of the compounds of this invention are acidic and they form a salt with a pharmaceutically acceptable cation. All such salts are within the scope of this invention and they can be prepared by conventional methods. For example, they can be prepared simply by contacting the acidic and basic entities, usually in a stoichiometric ratio, in either an aqueous, non-aqueous or partially aqueous medium, as appropriate. The salts are recovered either by filtration, by precipitation with a non-solvent followed by filtration, by evaporation of the solvent, or, in the case of aqueous solutions, by lyophilization, as appropriate.
In addition, some of the compounds of this invention are basic, and they form a salt with a pharmaceutically acceptable anion. All such salts are within the scope of this invention and they can be prepared by conventional methods. For example, they can be prepared simply by contacting the acidic and basic entities, usually in a stoichiometric ratio, in either an aqueous, non-aqueous or partially aqueous medium, as appropriate. The salts are recovered either by filtration, by precipitation with a non-solvent followed by filtration, by evaporation of the solvent, or, in the case of aqueous solutions, by lyophilization, as appropriate. In addition, when the compounds of this invention form hydrates or solvates they are also within the scope of the invention.
The utility of the compounds of the present invention as medical agents in the treatment of metabolic diseases (such as are detailed herein) in mammals (e.g. humans) is demonstrated by the activity of the compounds of this invention in conventional assays and the in vitro and in vivo assays described below. Such assays also provide a means whereby the activities of the compounds of this invention can be compared with the activities of other known compounds. The results of these comparisons are useful for determining dosage levels in mammals, including humans, for the treatment of such diseases.
The purified human liver glycogen phosphorylase a (HLGPa) is obtained by the following procedure.
Expression and fermentation:
The HLGP cDNA is expressed from plasmid pKK233-2 (Pharmacia Biotech. Inc., Piscataway, N.J.) in E. coli strain XL-1 Blue (Stratagene Cloning Systems, LaJolla, Calif.). The strain is inoculated into LB medium (consisting of 10 g tryptone, 5 g yeast extract, 5 g NaCl, and 1 ml 1N NaOH per liter) plus 100 mg/L ampicillin, 100 mg/L pyridoxine and 600 mg/L MnCl2 and grown at 37xc2x0 C. to a cell density of OD550=1.0. At this point, the cells are induced with 1 mM isopropyl-1-thio-xcex2-D-galactoside (IPTG). Three hours after induction the cells are harvested by centrifugation and cell pellets are frozen at xe2x88x9270xc2x0 C. until needed for purification.
Purification of Glycogen PhosDhorylase:
The cells in pellets described above are resuspended in 25 mM xcex2-glycerophosphate (pH 7.0) with 0.2 mM DTT, 1 mM MgCl2, plus the following protease inhibitors:
lysed by pretreatment with 200 xcexcg/mL lysozyme and 3 xcexcg/mL DNAase followed by sonication in 250 mL batches for 5xc3x971.5 minutes on ice using a Branson Model 450 ultrasonic cell disrupter (Branson Sonic Power Co., Danbury Conn.). The lysates are cleared by centrifugation at 35,000xc3x97g for one hour followed by filtration through 0.45 micron filters. HLGP in the soluble fraction of the lysates (estimated to be less than 1% of the total protein) is purified by monitoring the enzyme activity (as described in HLGPa Activity Assay section, below) from a series of chromatographic steps detailed below.
Immobilized Metal Affinity Chromatography (IMAC):
This step is based on the method of Luong et al (Luong et al. Journal of Chromatography (1992) 584, 77-84.). 500 mL of the filtered soluble fraction of cell lysates (prepared from approximately 160 g of original cell pellet) are loaded onto a 130 mL column of IMAC Chelating-Sepharose (Pharmacia LKB Biotechnology, Piscataway, N.J.) which has been charged with 50 mM CuCl2 and 25 mM xcex2-glycerophosphate, 250 mM NaCl and 1 mM imidazole at pH 7 equilibration buffer. The column is washed with equilibration buffer until the A280 returns to baseline. The sample is then eluted from the column with the same buffer containing 100 mM imidazole to remove the bound HLGP and other bound proteins. Fractions containing the HLGP activity are pooled (approximately 600 mL), and ethylenediaminetetraacetic acid (EDTA), DL-dithiothreitol (DTT), phenylmethylsulfonyl fluoride (PMSF), leupeptin and pepstatin A are added to obtain 0.3 mM, 0.2 mM, 0.2 mM, 0.5 xcexcg/mL and 0.7 xcexcg/mL concentrations respectively. The pooled HLGP is desalted over a Sephadex G-25 column (Sigma Chemical Co., St. Louis, Mo.) equilibrated with 25 mM Tris-HCl (pH 7.3), 3 mM DTT buffer (Buffer A) to remove imidazole and is stored on ice until the second chromatographic step.
5xe2x80x2- AMP-Sepharose Chromatography:
The desalted pooled HLGP sample (approximately 600 mL) is next mixed with 70 mL of 5xe2x80x2-AMP Sepharose (Pharmacia LKB Biotechnology, Piscataway, N.J.) which has been equilibrated with Buffer A (see above). The mixture is gently agitated for one hour at 22xc2x0 C. then packed into a column and washed with Buffer A until the A280 returns to baseline. HLGP and other proteins are eluted from the column with 25 mM Tris-HCl, 0.2 mM DTT and 10 mM adenosine 5-monophosphate (AMP) at pH 7.3 (Buffer B). HLGP-containing fractions are pooled following identification by determining enzyme (described below) activity and visualizing the M, approximately 97 kdal HLGP protein band by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) followed by silver staining (2D-silver Stain II xe2x80x9cDalichi Kitxe2x80x9d, Daiichi Pure Chemicals Co., LTD., Tokyo, Japan) and then pooled. The pooled HLGP is dialyzed into 25 mM xcex2-glycerophosphate, 0.2 mM DTT, 0.3 mM EDTA, 200 mM NaCl, pH 7.0 buffer (Buffer C) and stored on ice until use.
Determination of HLGP Enzyme Activity:
A) Activation of HLGP: Conversion of HLGPb to HLGPa
Prior to the determination of HLGP enzyme activity, the enzyme is converted from the inactive form as expressed in E. coli strain XL-1 Blue (designated HLGPb) (Stragene Cloning Systems, La Jolla, Calif.) to the active form (designated HLGPa) by phosphorylation of HLGP using phosphorylase kinase as follows:
HLGPb reaction with Immobilized Phosphorylase Kinase
Phosphorylase kinase (Sigma Chemical Company, St. Louis, Mo.) is immobilized on Affi-Gel 10 (BioRad Corp., Melvile, N.Y.) as per the manufacturer""s instructions. In brief, the phosphorylase kinase enzyme (10 mg) is incubated with washed Affi-Gel beads (1 mL) in 2.5 mL of 100 mM HEPES and 80 mM CaCl2 at pH 7.4 for 4 hours at 4xc2x0 C. The Affi-Gel beads are then washed once with the same buffer prior to blocking with 50 mM HEPES and 1 M glycine methyl ester at pH 8.0 for one hour at room temperature. Blocking buffer is removed and replaced with 50 mM HEPES (pH 7.4), 1 mM xcex2-mercaptoethanol and 0.2% NaN3 for storage. Prior to use to convert HLGPb to HLGPa, the Affi-Gel immobilized phosphorylase kinase beads are equilibrated by washing in the buffer used to perform the kinase reaction, consisting of 25 mM xcex2-glycerophosphate, 0.3 mM DTT, and 0.3 mM EDTA at pH 7.8 (kinase assay buffer).
The partially purified, inactive HLGPb obtained from 5xe2x80x2-AMP-Sepharose chromatography above is diluted 1:10 with the kinase assay buffer then mixed with the aforementioned phosphorylase kinase enzyme immobilized on the Affi-Gel beads. NaATP is added to 5 mM and MgCl2 to 6 mM. The resulting mixture is mixed gently at 25xc2x0 C. for 30 to 60 minutes. The sample is removed from the beads and the percent activation of HLGPb by conversion to HLGPa is estimated by determining HLGP enzyme activity in the presence and absence of 3.3 mM AMP. The percentage of total HLGP enzyme activity due to HLGPa enzyme activity (AMP-independent) is then calculated as follows:       %    ⁢          xe2x80x83        ⁢    of    ⁢          xe2x80x83        ⁢    total    ⁢          xe2x80x83        ⁢    HLGP    ⁢          xe2x80x83        ⁢    as    ⁢          xe2x80x83        ⁢          HLGP      a        =                    HLGP        ⁢                  xe2x80x83                ⁢        activity            -      AMP                      HLGP        ⁢                  xe2x80x83                ⁢        activity            +      AMP      
B) HLGPa Activity Assay:
The hypoglycemic activity (also the other disease/condition treating/preventing activities described herein) of the compounds of this invention can be indirectly determined by assessing the effect of the compounds of this invention on the activity of the activated form of glycogen phosphorylase (GPa) by one of two methods; glycogen phosphorylase a activity is measured in the forward direction by monitoring the production of glucose-1-phosphate from glycogen or by following the reverse reaction, measuring glycogen synthesis from glucose-1-phosphate by the release of inorganic phosphate. All reactions are run in triplicate in 96-well microtiter plates and the change in absorbance due to formation of the reaction product is measured at the wavelength specified below in a MCC/340 MKII Elisa Reader (Lab Systems, Finland), connected to a Titertech Microplate Stacker (ICN Biomedical Co, Huntsville, Ala.).
To measure the HLGPa enzyme activity in the forward direction, the production of glucose-1-phosphate from glycogen is monitored by the multienzyme coupled general method of Pesce et al. [Pesce, M. A., Bodourian, S. H., Harris, R. C. and Nicholson, J. F. (1977) Clinical Chemistry 23, 1711-1717] modified as follows: 1 to 100 xcexcg phosphorylase a, 10 units phosphoglucomutase and 15 units glucose-6-phosphate dehydrogenase (Boehringer Mannheim Biochemicals, Indianapolis, Ind.) are diluted to 1 mL in Buffer A (described hereinafter). Buffer A is at pH 7.2 and contains 50 mM HEPES, 100 mM KCl, 2.5 mM ethyleneglycoltetraacetic acid (EGTA), 2.5 mM MgCl2, 3.5 mM KH2PO4 and 0.5 mM dithiothreitol. 20 xcexcl of this stock is added to 80 xcexcl of Buffer A containing 0.47 mg/mL glycogen, 9.4 mM glucose, 0.63 mM of the oxidized form of nicotinamide adenine dinucleotide phosphate (NADP+). The compounds to be tested are added as 5 xcexcL of solution in 14% dimethylsulfoxide (DMSO) prior to the addition of the enzymes. The basal rate of HLGPa enzyme activity in the absence of inhibitors is determined by adding 5 xcexcL of 14% DMSO and a fully-inhibited rate of HLGPa enzyme activity is obtained by adding 20 xcexcL of 50 mM of the positive control test substance, caffeine. The reaction is followed at room temperature by measuring the conversion of oxidized NADP+ to reduced NADPH at 340 nm.
To measure HLGPa enzyme activity in the reverse direction, the conversion of glucose-1-phosphate into glycogen plus inorganic phosphate is measured by the general method described by Engers et al. [Engers, H. D., Shechosky, S. and Madsen, N. B. (1970) Can. J. Biochem. 48, 746-754] modified as follows: 1 to 100 ug HLGPa is diluted to 1 mL in Buffer B (described hereinafter). Buffer B is at pH 7.2 and contains 50 mM HEPES, 100 mM KCl, 2.5 mM EGTA, 2.5 mM MgCl2 and 0.5 mM dithiothreitol. 20 xcexcL of this stock is added to 80 xcexcL of Buffer B with 1.25 mg/mL glycogen, 9.4 mM glucose, and 0.63 mM glucose-1-phosphate. The compounds to be tested are added as 5 xcexcL of solution in 14% DMSO prior to the addition of the enzyme. The basal rate of HLGPa enzyme activity in the absence of added inhibitors is determined by adding 5 xcexcL of 14% DMSO and a fully-inhibited rate of HLGPa enzyme activity is obtained by adding 20 xcexcL of 50 mM caffeine. This mixture is incubated at room temperature for 1 hour and the inorganic phosphate released from the glucose-1-phosphate is measured by the general method of Lanzetta et al. [Lanzetta, P. A., Alvarez, L. J., Reinach, P. S. and Candia, O. A. (1979) Anal. Biochem. 100, 95-97] modified as follows: 150 xcexcL of 10 mg/mL ammonium molybdate, 0.38 mg/mL malachite green in 1 N HCl is added to 100 xcexcL of the enzyme mix. After a 20 minute incubation at room temperature, the absorbance is measured at 620 nm.
The compounds of this invention are readily adapted to clinical use as hypoglycemic agents. The hypoglycemic activity of the compounds of this invention can be determined by the amount of test compound that reduces glucose levels relative to a vehicle without test compound in male ob/ob mice. The test also allows the determination of an approximate minimal effective dose (MED) value for the in vivo reduction of plasma glucose concentration in such mice for such test compounds.
Since the concentration of glucose in blood is closely related to the development of diabetic disorders, these compounds by virtue of their hypoglycemic action, prevent, arrest and/or regress diabetic disorders.
Five to eight week old male C57BL/6J-ob/ob mice (obtained from Jackson Laboratory, Bar Harbor, Me.) are housed five per cage under standard animal care practices. After a one week acclimation period, the animals are weighed and 25 microliters of blood are collected from the retro-orbital sinus prior to any treatment. The blood sample is immediately diluted 1:5 with saline containing 0.025% sodium heparin, and held on ice for metabolite analysis. Animals are assigned to treatment groups so that each group has a similar mean for plasma glucose concentration. After group assignment, animals are dosed orally each day for four days with the vehicle consisting of either: 1) 0.25% w/v methyl cellulose in water without pH adjustment; or 2) 0.1% Pluronic(copyright) P105 Block Copolymer Surfactant (BASF Corporation, Parsippany, N.J.) in 0.1% saline without pH adjustment. On day 5, the animals are weighed again and then dosed orally with the test compound or the vehicle alone. All drugs are administered in vehicle consisting of either: 1) 0.25% w/v methyl cellulose in water without pH adjustment; or 2) 10% DMSO/0.1% Pluronic(copyright) P105 (BASF Corporation, Parsippany, N.J.) in 0.1% saline without pH adjustment. The animals are then bled from the retro-orbital sinus three hours later for determination of blood metabolite levels. The freshly collected samples are centrifuged for two minutes at 10,000xc3x97g at room temperature. The supernatant is analyzed for glucose, for example, by the Abbott VP(trademark)(Abbott Laboratories, Diagnostics Division, Irving, Tex.) and VP Super System(copyright) Autoanalyzer (Abbott Laboratories, Irving, Tex.), using the A-Gent(trademark) Glucose-UV Test reagent system (Abbott Laboratories, Irving, Tex.) (a modification of the method of Richterich and Dauwalder, Schweizerische Medizinische Wochenschrift, 101, 860 (1971)) (hexokinase method) using a 100 mg/dL standard. Plasma glucose is then calculated by the equation:
Plasma glucose (mg/dL)=Sample valuexc3x975xc3x971.784=8.92xc3x97Sample value 
where 5 is the dilution factor and 1.784 is the plasma hematocrit adjustment (assuming the hematocrit is 44%).
The animals dosed with vehicle maintain substantially unchanged hyperglycemic glucose levels (e.g., greater than or equal to 250 mg/dL), animals treated with test compounds at suitable doses have significantly depressed glucose levels. Hypoglycemic activity of the test compounds is determined by statistical analysis (unpaired t-test) of the mean plasma glucose concentration between the test compound group and vehicle-treated group on day 5. The above assay carried out with a range of doses of test compounds allows the determination of an approximate minimal effective dose (MED) value for the in vivo reduction of plasma glucose concentration.
The compounds of this invention are readily adapted to clinical use as hyperinsulinemia reversing agents, triglyceride lowering agents and hypocholesterolemic agents. Such activity can be determined by the amount of test compound that reduces insulin, triglycerides or cholesterol levels relative to a control vehicle without test compound in male ob/ob mice.
Since the concentration of cholesterol in blood is closely related to the development of cardiovascular, cerebral vascular or peripheral vascular disorders, the compounds of this invention by virtue of their hypocholesterolemic action, prevent, arrest and/or regress atherosclerosis.
Since the concentration of insulin in blood is related to the promotion of vascular cell growth and increased renal sodium retention, (in addition to the other actions e.g., promotion of glucose utilization) and these functions are known causes of hypertension, the compounds of this invention by virtue of their hypoinsulinemic action, prevent, arrest and/or regress hypertension.
Since the concentration of triglycerides in blood contributes to the overall levels of blood lipids, the compounds of this invention by virtue of their triglyceride lowering activity prevent, arrest and/or regress hyperlipidemia.
Five to eight week old male C57BL/6J-ob/ob mice (obtained from Jackson Laboratory, Bar Harbor, Me.) are housed five per cage under standard animal care practices and fed standard rodent diet ad libitum. After a one week acclimation period, the animals are weighed and 26 microliters of blood are collected from the retro-orbital sinus prior to any treatment. The blood sample is immediately diluted 1:5 with saline containing 0.025% sodium heparin, and held on ice for plasma glucose analysis. Animals are assigned to treatment groups so that each group has a similar mean for plasma glucose concentration. The compound to be tested is administered by oral gavage as an about 0.02% to 2.0% solution (weight/volume (w/v)) in either 1) 10% DMSO/0.1% Pluronic(copyright) P105 Block Copolymer Surfactant (BASF Corporation, Parsippany, N.J.) in 0.1% saline without pH adjustment or 2) 0.25% w/v methylcellulose in water without pH adjustment. Single daily dosing (s.i.d.) or twice daily dosing (b.i.d.) is maintained for 1 to 15 days. Control mice receive the 10% DMSO/0.1% Pluronic(copyright) P105 in 0.1% saline without pH adjustment or the 0.25% w/v methylcellulose in water without pH adjustment only.
Three hours after the last dose is administered, the animals are sacrificed by decapitation and trunk blood is collected into 0.5 mL serum separator tubes containing 3.6 mg of a 1:1 weight/weight sodium fluoride: potassium oxalate mixture. The freshly collected samples are centrifuged for two minutes at 10,000xc3x97g at room temperature, and the serum supernatant is transferred and diluted 1:1 volume/volume with a 1TIU/mL aprotinin solution in 0.1% saline without pH adjustment.
The diluted serum samples are then stored at xe2x88x9280xc2x0 C. until analysis. The thawed, diluted serum samples are analyzed for insulin, triglycerides, and cholesterol levels. Serum insulin concentration is determined using Equate(copyright) RIA INSULIN kits (double antibody method; as specified by the manufacturer) purchased from Binax, South Portland, Me. The inter assay coefficient of variation is xe2x89xa610%. Serum triglycerides are determined using the Abbott VP(trademark) and VP Super System(copyright) Autoanalyzer (Abbott Laboratories, Irving, Tex.), using the A-Gent(trademark) Triglycerides Test reagent system (Abbott Laboratories, Diagnostics Division, Irving, Tex.) (lipase-coupled enzyme method; a modification of the method of Sampson, et al., Clinical Chemistry 21, 1983 (1975)). Serum total cholesterol levels are determined using the Abbott VP(trademark) and VP Super System(copyright) Autoanalyzer (Abbott Laboratories, Irving, Tex.), and A-Gent(trademark) Cholesterol Test reagent system (cholesterol esterase-coupled enzyme method; a modification of the method of Allain, et al. Clinical Chemistry 20, 470 (1974)) using a 100 and 300 mg/dL standards. Serum insulin, triglycerides, and total cholesterol levels are then calculated by the equations,
Serum insulin (xcexcU/mL)=Sample valuexc3x972 
Serum triglycerides (mg/dL)=Sample valuexc3x972 
Serum total cholesterol (mg/dL)=Sample valuexc3x972 
where 2 is the dilution factor.
The animals dosed with vehicle maintain substantially unchanged, elevated serum insulin (e.g. 225 xcexcU/mL), serum triglycerides (e.g. 225 mg/dl), and serum total cholesterol (e.g. 160 mg/dL) levels, while animals treated with test compounds of this invention generally display reduced serum insulin, triglycerides, and total cholesterol levels. The serum insulin, triglycerides, and total cholesterol lowering activity of the test compounds are determined by statistical analysis (unpaired t-test) of the mean serum insulin, triglycerides, or total cholesterol concentration between the test compound group and the vehicle-treated control group.
Activity in providing protection from damage to heart tissue for the compounds of this invention can be demonstrated in vitro along the lines presented in Butwell et al., Am. J. Physiol., 264, H1884-H1889, 1993 and Allard et al., Am. J. Physio., 1994, 267, H66-H74. Experiments are performed using an isovolumic isolated rat heart preparation, essentially as described in the above-referenced article. Normal male Sprague-Dawley rats, male Sprague-Dawley rats treated to possess cardiac hypertrophy by an aortic banding operation, acutely diabetic male BB/W rats, or non-diabetic BB/W age matched control rats are pretreated with heparin (1000 u, i.p.), followed by pentobarbital (65 mg/kg, i.p.). After deep anesthesia is achieved as determined by the absence of a foot reflex, the heart is rapidly excised and placed into iced saline. The heart is retrogradely perfused through the aorta within 2 minutes. Heart rate and ventricular pressure are determined using a latex balloon in the left ventricle with high pressure tubing connected to a pressure transducer. The heart is perfused with a perfusate solution consisting of (mM) NaCl 118, KCl 4.7, CaCl2 1.2, MgCl2 1.2, NaHCO3 25, glucose 11. The perfusion apparatus is tightly temperature-controlled with heated baths used for the perfusate and for the water jacketing around the perfusion tubing to maintain heart temperature at 37xc2x0 C. Oxygenation of the perfusate is provided by a pediatric hollow fiber oxygenator (Capiax, Terumo Corp., Tokyo, Japan) immediately proximal to the heart. Hearts are exposed to perfusion solutionxc2x1test compound for about 10 minutes or more, followed by 20 minutes of global ischemia and 60 minutes of reperfusion in the absence of the test compound. The heart beats of the control and test compound treated hearts are compared in the period following ischemia. The left ventricular pressure of the control and test compound treated hearts are compared in the period following ischemia. At the end of the experiment, hearts are also perfused and stained to determine the ratio of infarct area relative to the area at risk (% IA/AAR) as described below.
The therapeutic effects of the compounds of this invention in preventing heart tissue damage otherwise resulting from an ischemic insult can also be demonstrated in vivo along lines presented in Liu et al., Circulation, Vol. 84, No. 1, (July 1991), as described specifically herein. The in vivo assay tests the cardioprotection of the test compound relative to the control group which receives saline vehicle. As background information, it is noted that brief periods of myocardial ischemia followed by coronary artery reperfusion protects the heart from subsequent severe myocardial ischemia (Murry et al., Circulation 74:1124-1136, 1986). Cardioprotection, as indicated by a reduction in infarcted myocardium, can be induced pharmacologically using intravenously administered adenosine receptor agonists in intact, anesthetized rabbits studied as an in situ model of myocardial ischemic preconditioning (Liu et al., Circulation 84:350-356, 1991). The in vivo assay tests whether compounds can pharmacologically induce cardioprotection, i.e., reduced myocardial infarct size, when parenterally administered to intact, anesthetized rabbits. The effects of the compounds of this invention can be compared to ischemic preconditioning using the A1 adenosine agonist, N6-1-(phenyl-2R-isopropyl) adenosine (PIA) that has been shown to pharmacologically induce cardioprotection in intact anesthetized rabbits studied in situ (Lu et al., Circulation 84:350-356, 1991). The exact methodology is described below.
Surgery: New Zealand White male rabbits (3-4 kg) are anesthetized with sodium pentobarbital (30 mg/kg, i.v.). A tracheotomy is performed via a ventral midline cervical incision and the rabbits are ventilated with 100% oxygen using a positive pressure ventilator. Catheters are placed in the left jugular vein for drug administration and in the left carotid artery for blood pressure measurements. The hearts are then exposed through a left thoracotomy and a snare (00 silk) placed around a prominent branch of the left coronary artery. Ischemia is induced by pulling the snare tight and clamping it in place. Releasing the snare allowed the affected area to reperfuse. Myocardial ischemia is evidenced by regional cyanosis; reperfusion was evidenced by reactive hyperemia.
Protocol: Once arterial pressure and heart rate has been stable for at least 30 minutes the experiment is started. Ischemic preconditioning is induced by twice occluding the coronary artery for 5 min followed by a 10 min reperfusion. Pharmacological preconditioning is induced by twice infusing test compound over, for example 5 minutes and allowing 10 minutes before further intervention or by infusing the adenosine agonist, PIA (0.25 mg/kg). Following ischemic preconditioning, pharmacological preconditioning or no conditioning (unconditioned, vehicle control) the artery is occluded for 30 minutes and then reperfused for two hours to induce myocardial infarction. The test compound and PIA are dissolved in saline or other suitable vehicle and delivered at 1 to 5 ml/kg, respectively.
Staining (Liu et al., Circulation 84:350-356, 1991): At the end of the 2 hour reperfusion period, the hearts are quickly removed, hung on a Langendorff apparatus, and flushed for 1 minute with normal saline heated to body temperature (38xc2x0 C.). The silk suture used as the snare is then tied tightly to reocclude the artery and a 0.5% suspension of fluorescent particles (1-10 xcexcm) is infused with the perfusate to stain all of the myocardium except the area at risk (nonfluorescent ventricle). The hearts are then quickly frozen and stored overnight at xe2x88x9220xc2x0 C. On the following day, the hearts are cut into 2 mm slices and stained with 1% triphenyl tetrazolium chloride (TTC). Since TTC reacts with living tissue, this stain differentiates between living (red stained) tissue, and dead tissue (unstained infarcted tissue). The infarcted area (no stain) and the area at risk (no fluorescent particles) are calculated for each slice of left ventricle using a pre-calibrated image analyzer. To normalize the ischemic injury for differences in the area at risk between hearts, the data is expressed as the ratio of infarct area vs. area at risk (% IA/AAR). All data is expressed as Meanxc2x1SEM and compared statistically using single factor ANOVA or unpaired t-test. Significance is considered as p less than 0.05.
Administration of the compounds of this invention can be via any method which delivers a compound of this invention preferentially to the liver and/or cardiac tissues. These methods include oral routes, parenteral, intraduodenal routes, etc. Generally, the compounds of the present invention are administered in single (e.g., once daily) or multiple doses.
However, the amount and timing of compound(s) administered will, of course, be dependent on the particular disease/condition being treated, the subject being treated, on the severity of the affliction, on the manner of administration and on the judgment of the prescribing physician. Thus, because of patient to patient variability, the dosages given below are a guideline and the physician may titrate doses of the drug to achieve the activity (e.g., glucose lowering activity) that the physician considers appropriate for the patient. In considering the degree of activity desired, the physician must balance a variety of factors such as starting level, other risk (cardiovascular) factors, presence of preexisting disease, and age of the patient and the patient""s motivation.
In general an effective dosage for the activities of this invention, for example the blood glucose, triglycerides, and cholesterol lowering activities and hyperinsulinemia reversing activities of the compounds of this invention is in the range of 0.005 to 50 mg/kg/day, preferably 0.01 to 25 mg/kg/day and most preferably 0.1 to 15 mg/kg/day.
Generally, the compounds of this invention are administered orally, but parenteral administration (e.g., intravenous, intramuscular, subcutaneous or intramedullary) may be utilized, for example, where oral administration is inappropriate for the instant target or where the patient is unable to ingest the drug. Topical administration may also be indicated, for example, where the patient is suffering from gastrointestinal disorders or whenever the medication is best applied to the surface of a tissue or organ as determined by the attending physician.
The compounds of the present invention are generally administered in the form of a pharmaceutical composition comprising at least one of the compounds of this invention together with a pharmaceutically acceptable vehicle or diluent. Thus, the compounds of this invention can be administered individually or together in any conventional oral, parenteral or transdermal dosage form.
For oral administration a pharmaceutical composition can take the form of solutions, suspensions, tablets, pills, capsules, powders, and the like. Tablets containing various excipients such as sodium citrate, calcium carbonate and calcium phosphate are employed along with various disintegrants such as starch and preferably potato or tapioca starch and certain complex silicates, together with binding agents such as polyvinylpyrrolidone, sucrose, gelatin and acacia. Additionally, lubricating agents such as magnesium stearate, sodium lauryl sulfate and talc are often very useful for tabletting purposes. Solid compositions of a similar type are also employed as fillers in soft and hard-filled gelatin capsules; preferred materials in this connection also include lactose or milk sugar as well as high molecular weight polyethylene glycols. When aqueous suspensions and/or elixirs are desired for oral administration, the compounds of this invention can be combined with various sweetening agents, flavoring agents, coloring agents, emulsifying agents and/or suspending agents, as well as such diluents as water, ethanol, propylene glycol, glycerin and various like combinations thereof.
For purposes of parenteral administration, solutions in sesame or peanut oil or in aqueous propylene glycol can be employed, as well as sterile aqueous solutions of the corresponding water-soluble salts. Such aqueous solutions may be suitably buffered, if necessary, and the liquid diluent first rendered isotonic with sufficient saline or glucose. These aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal injection purposes. In this connection, the sterile aqueous media employed are all readily obtainable by standard techniques well-known to those skilled in the art.
For purposes of transdermal (e.g., topical) administration, dilute sterile, aqueous or partially aqueous solutions (usually in about 0.1% to 5% concentration), otherwise similar to the above parenteral solutions, are prepared.
Methods of preparing various pharmaceutical compositions with a certain amount of active ingredient are known, or will be apparent in light of this disclosure, to those skilled in this art. For examples, see Remington""s Pharmaceutical Sciences, Mack Publishing Company, Easter, Pa., 15th Edition (1975).
Pharmaceutical compositions according to the invention may contain 0.1%-95% of the compound(s) of this invention, preferably 1%-70%. In any event, the composition or formulation to be administered will contain a quantity of a compound(s) according to the invention in an amount effective to treat the disease/condition of the subject being treated, i.e., a glycogen phosphorylase dependent disease/condition.
NMR spectra were recorded on a Varian XL-300 (Varian Co., Palo Alto, Calif.) or Bruker AM-300 spectrometer (Bruker Co., Billerica, Mass.) at about 23xc2x0 C. at 300 MHz for proton and 75.4 mHz for carbon nuclei. Chemical shifts are expressed in parts per million downfield from trimethylsilane. Resonances designated as exchangeable did not appear in a separate NMR experiment where the sample was shaken with several drops of D2O in the same solvent. FAB-MS spectra were obtained on a VG70-2505 spectrometer (V4 analytical LTD., Wythanshaw, Manchester, U.K.) using a liquid matrix consisting of 3:1 dithiothreitol/dithioerythritol. Thermospray MS (TSPMS) were obtained on a Fisons Trio-1000 spectrometer (Fisons Co., Valencia, Calif.) using ammonia ionization. Chemical ionization mass spectra were obtained on a Hewlett-Packard 5989 *n instrument (Hewlett-Packard Co., Palo Alto, Calif.) (ammonia ionization, PBMS). Where the intensity of chlorine or bromine-containing ions are described the expected intensity ratio was observed (approximately 3:1 for 35Cl/37Cl-containing ions) and 1:1 for 79Br/81Br-containing ions) and the intensity of only the lower mass ion is given.
HPLC was performed with 214 nM detection on a 250xc3x974.6 mm Rainin Microsorb C-18 column (Rainin Co., Wobum, Mass.) eluted isocratically by a two-pump/mixer system supplying the indicated mixture of acetonitrile and aqueous pH 2.1 (with H3PO4) 0.1M KH2PO4, respectively, at 1.5 mL/min. Samples were injected in a 1:1 mixture of acetonitrile and pH 7.0 phosphate buffer (0.025M in each Na2HPO4 and KH2PO4). Percent purities refer to percent of total integrated area usually over a 10 to 15 minute run. Melting points are uncorrected and were determined on a Buchi 510 melting point apparatus (Buchi Laboratorums-Technik Ag., Flawil, Switzerland) where melting points of 120.5-122xc2x0 C. for benzoic acid and 237.5-240.5xc2x0 C. for p-chlorobenzoic acid (Aldrich 99+% grades) were obtained. Column chromatography was performed with Amicon silica gel (30 uM, 60A pore size) (Amicon D Vision, W. R. Grace and Co., Beverly, Mass.) in glass columns under low nitrogen pressure. Unless otherwise specified, reagents were used as obtained from commercial sources. Dimethylformamide, 2-propanol, tetrahydrofuran, and dichloromethane used as reaction solvents were the anhydrous grade supplied by Aldrich Chemical Company (Milwaukee, Wis.). Microanalyses were performed by Schwarzkopf Microanalytical Laboratory, Woodside, N.Y. The terms xe2x80x9cconcentratedxe2x80x9d and coevaporated refer to removal of solvent at water aspirator pressure on a rotary evaporator with a bath temperature of less than 45xc2x0 C.
An 0.1-0.7 M solution of the primary amine (1.0 equiv, or a primary amine hydrochloride and 1.0 to 1.3 equivalents of triethyl amine per equiv HCl) in dichloromethane (unless other solvent specified), is treated sequentially at 25xc2x0 C. with 0.95 to 1.2 equivalent of the specified carboxylic acid, 1.2 to 1.8 equivalent hydroxybenzotriazole hydrate (usually 1.5 equivalent relative to the carboxylic acid), and 0.95-1.2 equivalent (corresponding in mole ratio to the carboxylic acid) 1-(3-dimethylaminopropyl)3-ethylcarbodiimide hydrochloride (DEC) and the mixture is stirred for 14 to 20 hours. (See Note 1 below). The mixture is diluted with ethyl acetate, washed 2 to 3 times with 1 or 2N NaOH, 2 to 3 times with 1 or 2N HCl (Note 2), the organic layer dried over MgSO4, and concentrated giving crude product which is purified by chromatography on silica gel, trituration, or recrystallization, as specified using the specified solvents. Purified products were analyzed by RP-HPLC and found to be of greater than 95% purity unless otherwise noted. Exceptions in the use of Procedure A are noted individually where appropriate below. Reactions conducted at 0 to 25xc2x0 C. were conducted with initial cooling of the vessel in an insulated ice bath which was allowed to warm to room temperature over several hours.
Note 1: On larger scale couplings ( greater than 50 mL solvent) the mixture was concentrated at this point and the residue dissolved in ethyl acetate. Note 2: If the product contained ionizable amine functionality the acid wash was omitted.